Assessment of risk of hypertension and methods based thereon

ABSTRACT

Provided is a method of analysing whether the blood pressure of a subject is sensitive to sodium intake and/or of assessing the susceptibility of a subject to develop hypertension. In the method erythrocytes of the subject are suspended in two sodium chloride solutions of about physiological osmolality. The sodium chloride concentration of one of the two solutions is at least about 25 mM lower than in the other solution. After allowing the suspended erythrocytes in the two sodium chloride solutions to settle for a period of time sufficient to allow the formation of a supernatant the difference in height of the supernatant between the two sodium chloride solutions is detected. An increased difference in height of the supernatants, relative to a threshold value, indicates that the subject&#39;s blood pressure is sensitive to sodium intake and/or that the subject is susceptible to develop hypertension.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of and the priority to anapplication for “Assessment of Risk of Hypertension and Methods BasedThereon” filed on 27 Nov. 2012 with the European Patent Office, andthere duly assigned serial number EP 12 194 442. The content of saidapplication filed on 27 Nov. 2012 is incorporated herein by referencefor all purposes in its entirety including all tables, figures, andclaims—as well as including an incorporation of any element or part ofthe description, claims or drawings not contained herein and referred toin Rule 20.5(a) of the PCT, pursuant to Rule 4.18 of the PCT.

FIELD OF THE INVENTION

The present invention relates to the assessment of risk of hypertension,i.e. assessing the risk of occurrence of hypertension in a subject. Theinvention also relates to methods based on such risk assessment.Provided is also a method of diagnosing an increased risk of developingarterial hypertension and/or of stratifying the risk of developingarterial hypertension, as well as a method of determining whether theblood pressure of a subject is sensitive to sodium intake.

BACKGROUND OF THE INVENTION

The following description is provided to assist the understanding of thereader. None of the information provided or references cited is admittedto be prior art to the present invention.

Circulatory disorders are worldwide the number one cause of death. Theirmain cause is the wear-out of arterial blood vessels over the course oflife. Late effects of impaired vessels are stroke (apoplexy) and cardiacinfarction. Hypertension (arterial hypertension) is regarded the mostimportant factor in the pathogenesis of cardiovascular disorders(Meneton, P, et al., Physiol Rev (2005) 85, 679-715). With more thaneight billion Euro per year alone in Germany, as well as the resultingfollow-up costs the treatment of hypertension is the most expensive ofall diseases. Almost half of the worldwide population above 50 years ofage suffers from hypertension and will, with high likelihood, sufferfrom the long-term effects. The main difficulty in this regard is earlydiagnosis.

Hypertension is diagnosed when systolic blood pressure values areconsistently above 140 mm Hg and/or diastolic blood pressure values areabove 90 mm Hg. It is recommended that individuals with values in therange of 120-139/80-89 be categorized as having prehypertension(Sutters, M, in: Current Medical Diagnosis and Treatment (2013), 52ndedition McGraw Hill, Ch 11). 50% of individuals with prehypertensiondevelop hypertension within four years.

Complications associated with manifest hypertension are irreversiblealterations of the vasculature and the heart as well as atherosclerosisconnected to long lasting hypertension. Chronic hypertension furtherleads to nephrosclerosis. At the stage of irreversible alterations only“damage control” (by lifelong application of drugs, e.g.,antihypertensives), can be done, but no healing as such is achievable.The onset of essential hypertension is generally at an age from 25 to 55years. Only for about 5% of patients with hypertension a specific causecan be identified such as a drug-induced effect or a chronic kidneydisease. Unfortunately only repeated measurements of the arterial bloodpressure reveals existing (manifest) hypertension, so that “arterialhypertension” is only diagnosed at a stage where the blood vessels inall likelihood are already irreversibly damaged (proliferation of theconnective tissue, rarefication of elastic fibers of the main arteries,hypertrophy of vascular smooth muscle).

European patent EP 2 215 480 B1 discloses that perturbation of theendothelial glycocalyx can be diagnosed in a non-invasive manner by asize distribution method as well as on the basis of glycocalyx markers,and that this analysis is useful in the diagnosis of vascular diseases.

The search for possibilities of the early diagnosis of a “potentialhypertension” is of central interest, inter alia, because of itsenormous economic relevance. Unfortunately early expectations that itmight be possible to predict development of hypertension via a “geneticfingerprint” have not been fulfilled, as numerous studies on the humangenome over the last two decades have shown (Lupton, S. J., et al., TwinRes Hum Genet (2011) 14, 295-304). Hypertension cannot be ascribed to afew dysfunctional genes or their respective gene products.

Over the past years clinical detection methods such as the method ofpulse wave analysis (Gurovich A N & Braith R W, Hypertens Res (2011) 34,166-169) or various imaging technologies (e.g. intima media thickness)have been refined to a degree that the structural/functional state ofthe vascular system can be conceived in detail. Apart from the relativecomplexity of these examinations, which does not permit a widespread usethroughout the population for purely preventive purposes, these modernmethods do only detect the state of the blood vessels once alreadyvisible (structural) changes have occurred. Therefore, these methods arenot suitable for the early diagnosis of a disposition for hypertension.

The onset of hypertension is thought to be directly linked tointake/clearance of sodium chloride and water. The daily sodium intakein industrial countries exceeds physiological needs to a high degree.While for millions of years the evolutionary ancestors of modern man hada diet containing less than 1 g salt/day, present consumption is in therange of about 10 g salt/day. It is generally accepted that the highamount of sodium consumed via modern nutrition (rich in salt), includingfinished products, often overburdens the excretion capacity of thekidneys. As a result sodium is kept and stocked in the body and in thecourse of years blood vessels (Oberleithner, H., et al., Proc Natl AcadSci USA (2007), 104, 16281-16286) damages to organs such as the brain,kidneys and the heart (Oberleithner, H, & de Wardener, H. E., BloodPurif (2011) 31, 82-85).

Though efforts are made worldwide to limit sodium consumption (see WHOreport:http://www.who.int/dietphysicalactivity/reducingsalt/en/index.html), butthis development (reduction of the everyday sodium consumption by atleast 50%) will occur only very slowly, if at all. Thus the search formeans of early diagnosis of a potential hypertension, i.e. ofidentifying an increased risk of developing hypertension, is of centralinterest, inter alia due to its tremendous economic importance (supra).

It would therefore be advantageous to have available means that allowthe diagnosis of an increased risk of developing hypertension. It wouldbe particularly advantageous if such means would allow carrying out aquick and easy test for a respective diagnosis.

SUMMARY OF THE INVENTION

The present invention provides a method, a kit and a use of such kitthat can be used for a quick and simple test in medical diagnosis aswell as in determining whether the blood pressure of a subject issensitive to sodium intake, in particular whether the blood pressure ofa subject has an increased sensitivity to sodium intake. Arterialhypertension and related vascular diseases have high mortality ratesworldwide. A major risk factors in this regard is high sodium intake. Aresulting imbalance between sodium intake and renal sodium excretionresults in an increased risk that the subject will develop hypertension.Sodium sensitivity, i.e., the development of hypertension in response toa sodium salt, in particular NaCl, differs among people. A method and akit described herein can be used to determine this sodium sensitivity ofan individual.

In the following a reference to a method is intended to include a kitdisclosed herein, and/or the use of such kit, as applicable. Typicallysuch a test can thereby be used to indicate whether the vascular systemof an individual has an increased sensitivity to sodium intake, i.e.whether the vascular system of an individual is more sensitive to sodiumintake than an average individual of the same age group. An individualwith increased sensitivity to sodium intake shows an increased responsein the form of rise in blood pressure. The test can be used to assess ata pre-hypertension state whether a subject will be in need ofhypertension therapy and can be used in prognosis and risk assessment.Hence, precautionary measures in terms of medical prevention can betaken already at a stage where no damage has occurred in the subject'sorgans and circulatory system. A method or use as disclosed in thisdocument provides quantitative data, can be performed rapidly and iseasy to carry out. A method/use described herein does not requireparticular expert knowledge and can be carried out withoutcost-intensive equipment.

A method as disclosed herein may include staging, monitoring,categorizing and/or determining a subject's risk of developinghypertension and/or a hypertension induced disease, as well as staging,monitoring, categorizing and/or determination of further diagnosis andtreatment regimens in a subject at risk of suffering from hypertension.

In a first aspect the present invention provides an in vitro method ofanalysing whether the blood pressure of a subject is sensitive to sodiumintake. The method further includes detecting at least one of (i) thethickness (height) or the volume of the glycocalyx of erythrocytes ofthe subject, and (ii) the zeta potential of the erythrocytes, i.e. thedegree of electrostatic repulsion between erythrocytes of the subject. Adecreased height or volume of the glycocalyx of the erythrocytes,relative to a threshold value, indicates that the blood pressure of thesubject is sensitive to sodium intake. Further, a decreased zetapotential of the erythrocytes, relative to a threshold value, indicatesthat the blood pressure of the subject is sensitive to sodium intake.

The method includes in some embodiments suspending erythrocytes of thesubject in a solution of an inorganic salt of about physiologicalosmolality. Suspending the erythrocytes may in some embodiments becarried out before detecting the thickness (height) or the volume of theglycocalyx of erythrocytes of the subject, and/or detecting the zetapotential of the erythrocytes.

In some embodiments the inorganic salt is a sodium salt such as sodiumchloride. In some embodiments a respective sodium salt is Na₂SO₄. Insome embodiments a respective sodium salt is Na₂NO₃. In some embodimentsthe sodium salt is Na₂CO₃. In some embodiments the sodium salt isNa₂HPO₄ NaH₂PO₄ or Na₃PO₄. In some embodiments the inorganic salt is apotassium salt. Examples of a suitable potassium salt include, but arenot limited to, KCl, Ka₂NO₃ and K₂SO₄. In some embodiments the inorganicsalt is a calcium salt such as CaCl₃, Ca₃(PO₃)₂, Ca(NO₃)₂, CaCO₃ orCaCl₂. In some embodiments the inorganic salt is an aluminium salt, suchas AlCl₃ or Al₂(SO₄)₃. In some embodiments the inorganic salt is amagnesium salt such as MgCl₂ or MgSO₄. In some embodiments the inorganicsalt is a lithium salt. A suitable lithium salt may for example be LiClor Li₂SO₄. In some embodiments the inorganic salt is a zinc salt such asZnSO₄, ZnCl₂ or Zn(SO₄)₂. In some embodiments the inorganic salt is amixture of at least two of a sodium salt, a potassium salt, a calciumsalt, an aluminium salt, a lithium salt, a magnesium salt, an iron salt,a copper salt, a nickel salt and a zinc salt.

In some embodiments the height and/or the volume of the glycocalyx oferythrocytes is analysed by atomic force microscopy.

In some embodiments the zeta potential of the erythrocytes is determinedusing a zeta potential analyser. In some embodiments the zeta potentialis analysed by comparing the aggregation tendency of erythrocytes in twodifferent solutions, which both have an about physiological osmolality,but differ in the concentration of the inorganic salt. In such anembodiment a method according to the first aspect includes suspendingerythrocytes of the subject in a first solution of an inorganic salt.The first solution is of about physiological osmolality. The method ofsuch an embodiment also includes suspending erythrocytes of the subjectin a second solution of an inorganic salt. The second solution islikewise of about physiological osmolality. In some embodiments thefirst and the second solution are both solutions that contain sodiumchloride, which are at least essentially void of potassiumchloride—herein also referred to as a sodium chloride solution. If boththe first and the second solution are a sodium chloride solution, thefirst sodium chloride solution has a sodium chloride concentration thatis lower than the sodium chloride concentration of the second solution.In one embodiment the sodium chloride concentration in the first sodiumchloride solution is about 25 mM or more lower than the sodium chlorideconcentration of the second sodium chloride solution. In someembodiments comparing the aggregation tendency of erythrocytes in twodifferent solutions of an inorganic salt includes allowing theerythrocytes suspended in the two solutions of an inorganic salt tosettle over a period of time. In one embodiment where the inorganic saltis sodium chloride the method includes allowing the erythrocytessuspended in the first sodium chloride solution and the erythrocytessuspended in the second sodium chloride solution to settle over a periodof time. The suspended erythrocytes are allowed to settle for a periodof time that is sufficient to allow the formation of a supernatant. Inembodiments where two different solutions of an inorganic salt areemployed for analysing the aggregation tendency of erythrocytes themethod further includes detecting the difference in height of thesupernatant between the two solutions of an inorganic salt. Inembodiments where the inorganic salt is sodium chloride the methodgenerally includes detecting the difference in height of the supernatantbetween the first and the second sodium chloride solution. Generally thedifference in height of the supernatant can also be detected bydetecting the difference in height of the settled erythrocytes, i.e. theheight of the suspension that still contains erythrocytes, between thetwo solutions of the inorganic salt, such as the first and in the secondsodium chloride solution. An increased difference in height of thesupernatant between the two solutions of the inorganic salt, e.g.between the first and the second sodium chloride solution, relative to athreshold value, indicates that the blood pressure of the subject issensitive to sodium intake.

In some embodiments the method according to the first aspect includescomparing the height or volume of the glycocalyx of the erythrocytes toa threshold value, which may be a control measurement or a predeterminedreference value. In embodiments where the inorganic salt is sodiumchloride the method may include comparing the difference in height ofthe supernatant between the first and the second sodium chloridesolution to a control measurement or to a predetermined reference value.In some embodiments the threshold value is based on the difference inheight of the supernatant between a corresponding first and acorresponding second sodium chloride solution of erythrocytes of acontrol sample.

If a reduced height or volume of the glycocalyx of the erythrocytes,relative to a threshold value, and/or a decreased zeta potential of theerythrocytes is detected, a method according to the first aspect mayinclude stratifying the subject for monitoring blood pressure. Inembodiments where the inorganic salt is sodium chloride and an elevateddifference in height of the supernatant between the first and the secondsodium chloride solution has been detected, a method according to thefirst aspect may include stratifying the subject for monitoring bloodpressure.

In some embodiments of the method according to the first aspect theaggregation tendency of erythrocytes is compared in two pairs of twodifferent solutions, with all solutions having an about physiologicalosmolality. The first pair of solutions contains a first and a secondsodium chloride solution. The first and the second solution of thisfirst pair of solutions contain sodium chloride, but differ in theconcentration of sodium chloride. In one embodiment the sodium chlorideconcentration in the first solution is about 25 mM or more lower thanthe sodium chloride concentration of the second sodium chloride solutionof the first pair of solutions. The second pair of solutions contains afirst and a second sodium solution. The first solution of this secondpair of solutions contains sodium chloride. The second solution of thissecond pair of solutions contains an inorganic salt different fromsodium chloride and is at least essentially void of sodium chloride. Inone embodiment the inorganic salt included in the second solution of thesecond pair of solutions is a potassium salt such as potassium chloride.Erythrocytes of the subject are being suspended in the solutions of boththe first and the second pair of solutions. In one embodiment theconcentration of sodium chloride in the first solution of the first pairof solutions is at least essentially the same as the concentration ofthe different inorganic salt in the second solution of the second pairof solutions.

In some embodiments of the method according to the first aspect repeatedsessions of the method according to the first aspect are carried out.Sessions of the method may be carried out at certain, e.g.predetermined, time intervals.

In a second aspect the present invention provides an in vitro method ofassessing the risk of occurrence of hypertension in a subject. Themethod includes detecting at least one of (i) the thickness (height) orthe volume of the glycocalyx of erythrocytes of the subject, and (ii)the zeta potential of the erythrocytes, i.e. the degree of electrostaticrepulsion between erythrocytes of the subject. A decreased height orvolume of the glycocalyx of the erythrocytes, relative to a thresholdvalue, indicates that the blood pressure of the subject is sensitive tosodium intake. Further, a decreased zeta potential of the erythrocytes,relative to a threshold value, indicates that the blood pressure of thesubject is sensitive to sodium intake.

In some embodiments the method further includes suspending erythrocytesof the subject in a solution of an inorganic salt of about physiologicalosmolality.

In some embodiments the inorganic salt is a sodium salt such as sodiumchloride. In some embodiments a respective sodium salt is Na₂SO₄. Insome embodiments a respective sodium salt is Na₂NO₃. In some embodimentsthe sodium salt is Na₂CO₃. In some embodiments the sodium salt isNa₂HPO₄ NaH₂PO₄ or Na₃PO₄. In some embodiments the inorganic salt is apotassium salt. Examples of a suitable potassium salt include, but arenot limited to, KCl, Ka₂NO₃ and K₂SO₄. In some embodiments the inorganicsalt is a calcium salt such as CaCl₃, Ca₃(PO₃)₂, Ca(NO₃)₂, CaCO₃ orCaCl₂. In some embodiments the inorganic salt is an aluminium salt, suchas AlCl₃ or Al₂(SO₄)₃. In some embodiments the inorganic salt is amagnesium salt such as MgCl₂ or MgSO₄. In some embodiments the inorganicsalt is a lithium salt. A suitable lithium salt may for example be LiClor Li₂SO₄. In some embodiments the inorganic salt is a zinc salt such asZnSO₄, ZnCl₂ or Zn(SO₄)₂. In some embodiments the inorganic salt is amixture of at least two of a sodium salt, a potassium salt, a calciumsalt, an aluminium salt, a lithium salt, a magnesium salt, an iron salt,a copper salt, a nickel salt and a zinc salt.

In some embodiments the height and/or the volume of the glycocalyx oferythrocytes is analysed by atomic force microscopy.

In some embodiments the zeta potential of the erythrocytes is determinedusing a zeta potential analyser. In some embodiments the zeta potentialis analysed by comparing the aggregation tendency of erythrocytes in twodifferent solutions, which both have an about physiological osmolality,but differ in the concentration of the inorganic salt. In such anembodiment a method according to the second aspect includes suspendingerythrocytes of the subject in a first solution of an inorganic salt.The first solution is of about physiological osmolality. The method ofsuch an embodiment also includes suspending erythrocytes of the subjectin a second solution of an inorganic salt. The second solution is ofabout physiological osmolality. In some embodiments the first and thesecond solution are both solutions that contain sodium chloride, whichare at least essentially void of potassium chloride—herein also referredto as a sodium chloride solution. If both the first and the secondsolution are a sodium chloride solution, the first sodium chloridesolution has a sodium chloride concentration that is lower than thesodium chloride concentration of the second solution. In one embodimentthe sodium chloride concentration in the first sodium chloride solutionis about 25 mM or more lower than the sodium chloride concentration ofthe second sodium chloride solution. In some embodiments comparing theaggregation tendency of erythrocytes in two different solutions of aninorganic salt includes allowing the erythrocytes suspended in the twosolutions of an inorganic salt to settle over a period of time. In oneembodiment where the inorganic salt is sodium chloride the methodincludes allowing the erythrocytes suspended in the first sodiumchloride solution and the erythrocytes suspended in the second sodiumchloride solution to settle over a period of time. The suspendederythrocytes are allowed to settle for a period of time that issufficient to allow the formation of a supernatant. In embodiments wheretwo different solutions of an inorganic salt are employed for analysingthe aggregation tendency of erythrocytes the method further includesdetecting the difference in height of the supernatant between the twosolutions of an inorganic salt. In embodiments where the inorganic saltis sodium chloride the method generally includes detecting thedifference in height of the supernatant between the first and the secondsodium chloride solution. Generally the difference in height of thesupernatant can also be detected by detecting the difference in heightof the settled erythrocytes, i.e. the height of the suspension thatstill contains erythrocytes, between the two solutions of the inorganicsalt, such as the first and in the second sodium chloride solution. Anincreased difference in height of the supernatant between the twosolutions of the inorganic salt, e.g. between the first and the secondsodium chloride solution, relative to a threshold value, indicates thatthe blood pressure of the subject is sensitive to sodium intake.

In some embodiments the method according to the second aspect includescomparing the height or volume of the glycocalyx of the erythrocytes toa threshold value, which may be a control measurement or a predeterminedreference value. In embodiments where the inorganic salt is sodiumchloride and two suspensions of erythrocytes have been prepared, themethod may include comparing the difference in height of the supernatantbetween the first and the second sodium chloride solution to a controlmeasurement or to a predetermined reference value. In some embodimentsthe threshold value is based on the difference in height of thesupernatant between a corresponding first and a corresponding secondsodium chloride solution of erythrocytes of a control sample.

If a reduced height or volume of the glycocalyx of the erythrocytes,relative to a threshold value, and/or a decreased zeta potential of theerythrocytes is detected, a method according to the second aspect mayinclude stratifying the subject for monitoring blood pressure. Inembodiments where the inorganic salt is sodium chloride and an elevateddifference in height of the supernatant between the first and the secondsodium chloride solution has been detected, a method according to thesecond aspect may include stratifying the subject for monitoring bloodpressure.

In some embodiments of the method according to the second aspect theaggregation tendency of erythrocytes is compared in two pairs of twodifferent solutions, with all solutions having an about physiologicalosmolality. The first pair of solutions contains a first and a secondsodium chloride solution. The first and the second solution of thisfirst pair of solutions contain sodium chloride, but differ in theconcentration of sodium chloride. In one embodiment the sodium chlorideconcentration in the first solution is about 25 mM or more lower thanthe sodium chloride concentration of the second sodium chloride solutionof the first pair of solutions. The second pair of solutions contains afirst and a second sodium solution. The first solution of this secondpair of solutions contains sodium chloride. The second solution of thissecond pair of solutions contains an inorganic salt different fromsodium chloride and is at least essentially void of sodium chloride. Inone embodiment the inorganic salt included in the second solution of thesecond pair of solutions is a potassium salt such as potassium chloride.Erythrocytes of the subject are being suspended in the solutions of boththe first and the second pair of solutions. In one embodiment theconcentration of sodium chloride in the first solution of the first pairof solutions is at least essentially the same as the concentration ofthe different inorganic salt in the second solution of the second pairof solutions. In one embodiment the concentration of sodium chloride inthe first solution of the second pair of solutions is about 25 mM ormore lower than the concentration of the different inorganic salt in thesecond solution of the second pair of solutions.

In some embodiments of the method according to the second aspectrepeated sessions of the method according to the second aspect arecarried out. Sessions of the method may be carried out at certain, e.g.predetermined, time intervals.

In a third aspect the present invention provides an in vitro method ofanalysing whether the blood pressure of a subject is sensitive to sodiumintake. The method includes suspending erythrocytes of the subject in afirst sodium chloride solution of about physiological osmolality. Themethod also includes suspending erythrocytes of the subject in a secondsodium chloride solution of about physiological osmolality. The firstsodium chloride solution has a sodium chloride concentration, which hasa value that is about 25 mM or more below the value of the sodiumchloride concentration of the second sodium chloride solution. Themethod further includes allowing the erythrocytes suspended in the firstsodium chloride solution and the erythrocytes suspended in the secondsodium chloride solution to settle over a period of time. The suspendederythrocytes are allowed to settle for a period of time that issufficient to allow the formation of a supernatant. The method alsoincludes detecting the difference in height of the supernatant betweenthe first and the second sodium chloride solution. Generally thedifference in height of the supernatant can also be detected bydetection the difference in height of the settled erythrocytes, i.e. theheight of the suspension that still contains erythrocytes, between thefirst and in the second sodium chloride solution. An increaseddifference in height of the supernatant between the first and the secondsodium chloride solution, relative to a threshold value, indicates thatthe blood pressure of the subject is sensitive to sodium intake.

In some embodiments the method according to the third aspect includescomparing the difference in height of the supernatant between the firstand the second sodium chloride solution to a control measurement or to apredetermined reference value.

In some embodiments the threshold value is based on the difference inheight of the supernatant between a corresponding first and acorresponding second sodium chloride solution of erythrocytes of acontrol sample.

If an increased difference in height of the supernatant between thefirst and the second sodium chloride solution has been detected, amethod according to the third aspect may include stratifying the subjectfor monitoring blood pressure.

In some embodiments of the method according to the third aspect repeatedsessions of the method according to the third aspect are carried out.Sessions of the method may be carried out at certain, e.g.predetermined, time intervals.

In some embodiments the first sodium chloride solution has a sodiumchloride concentration that has a value of about 60 mM or more below thevalue of the sodium chloride concentration of the second sodium chloridesolution. In some embodiments the first sodium chloride solution has asodium chloride concentration of about 100 mM and the second sodiumchloride solution has a sodium chloride concentration of about 125 mM.In some embodiments the first sodium chloride solution has a sodiumchloride concentration of about 80 mM and the second sodium chloridesolution has a sodium chloride concentration of about 140 mM.

In some embodiments of the method according to the third aspect thefirst and the second sodium chloride solution include a polysaccharide,for example in an amount of about 3% (w/w). If the polysaccharide isdextran, it typically has an average molecular weight of about 70,000Dalton. Where another polysaccharide is included in the first and thesecond sodium chloride solution, it may also have an average molecularweight of about 70,000 Dalton. In some embodiments the polysaccharidehas a molecular weight of about 70,000 Dalton.

In some embodiments one or both of the first and the second sodiumchloride solution include a monosaccharide or a disaccharide, such assucrose.

In some embodiments the first sodium chloride solution has a sodiumchloride concentration of about 125 mM, includes about 3% (w/w) dextranof a molecular weight of about 70,000 Dalton and 50 mM sucrose. In someof these embodiments the second sodium chloride solution has a sodiumchloride concentration of about 150 mM and includes about 3% (w/w)dextran of a molecular weight of about 70,000 Dalton. In one embodimentthe first sodium chloride solution is an aqueous solution that at leastessentially consists of (i) sodium chloride in a concentration of about110 mM, (ii) dextran of a molecular weight of about 70,000 Dalton in anamount of about 3% (w/w), and (iii) sucrose in a concentration of about80 mM. In this embodiment the second sodium chloride solution is anaqueous solution that at least essentially consists of (i) sodiumchloride in a concentration of about 130 mM (ii) dextran of a molecularweight of about 70,000 Dalton in an amount of about 3% (w/w), and (iii)sucrose in a concentration of about 40 mM. In one embodiment the firstsodium chloride solution is an aqueous solution that at leastessentially consists of (i) sodium chloride in a concentration of about125 mM, (ii) dextran of a molecular weight of about 70,000 Dalton in anamount of about 3% (w/w) and (iii) sucrose in a concentration of about50 mM. In this embodiment the second sodium chloride solution is anaqueous solution that at least essentially consists of (i) sodiumchloride in a concentration of about 150 mM and (ii) dextran of amolecular weight of about 70,000 Dalton in an amount of about 3% (w/w).

In a fourth aspect the present invention provides an in vitro method ofassessing the risk of occurrence of hypertension in a subject. Themethod includes suspending erythrocytes of the subject in a first sodiumchloride solution of about physiological osmolality. The method alsoincludes suspending erythrocytes of the subject in a second sodiumchloride solution of about physiological osmolality. The first sodiumchloride solution has a sodium chloride concentration, which has a valuethat is about 25 mM or more below the value of the sodium chlorideconcentration of the second sodium chloride solution. The method furtherincludes allowing the erythrocytes suspended in the first sodiumchloride solution and the erythrocytes suspended in the second sodiumchloride solution to settle over a period of time. The suspendederythrocytes are allowed to settle for a period of time that issufficient to allow the formation of a supernatant. The method alsoincludes detecting the difference in height of the supernatant betweenthe first and in the second sodium chloride solution. Generally thedifference in height of the supernatant can also be detected bydetection the difference in height of the settled erythrocytes, i.e. theheight of the suspension that still contains erythrocytes, between thefirst and in the second sodium chloride solution. An increased height ofthe supernatant relative to a threshold value, indicates an increasedrisk of occurrence of hypertension.

In some embodiments the method according to the fourth aspect is amethod of diagnosing and/or stratifying the risk of developinghypertension.

In some embodiments the method according to the fourth aspect includescomparing the difference in height of the supernatant between the firstand the second sodium chloride solution to a control measurement or to apredetermined reference value.

In some embodiments the threshold value is based on the difference inheight of the supernatant between a corresponding first and acorresponding second sodium chloride solution of erythrocytes of acontrol sample.

If an increased difference in height of the supernatant between thefirst and the second sodium chloride solution has been detected, amethod according to the fourth aspect may include stratifying thesubject for monitoring blood pressure.

In some embodiments the method of the fourth aspect further includesrepeatedly detecting the difference in height of the settlederythrocytes.

In some embodiments of the method according to the fourth aspect thefirst and the second sodium chloride solution include a polysaccharide,for example in an amount of about 3% (w/w). If the polysaccharide isdextran, it typically has an average molecular weight of about 70,000Dalton. Where a polysaccharide different from dextran is included in thefirst and the second sodium chloride solution, it may also have anaverage molecular weight of about 70,000 Dalton. In some embodiments thepolysaccharide has a molecular weight of about 70,000 Dalton.

In some embodiments one or both of the first and the second sodiumchloride solution include a monosaccharide or a disaccharide, such assucrose.

In some embodiments of the method according to the fourth aspect thefirst sodium chloride solution has a sodium chloride concentration ofabout 125 mM, includes about 3% (w/w) dextran of a molecular weight ofabout 70,000 Dalton and 50 mM sucrose. In some of these embodiments thesecond sodium chloride solution has a sodium chloride concentration ofabout 150 mM and includes about 3% (w/w) dextran of a molecular weightof about 70,000 Dalton. In one embodiment the first sodium chloridesolution is an aqueous solution that at least essentially consists of(i) sodium chloride in a concentration of about 125 mM, (ii) dextran ofa molecular weight of about 70,000 Dalton in an amount of about 3% (w/w)and (iii) sucrose in a concentration of about 50 mM. In this embodimentthe second sodium chloride solution is an aqueous solution that at leastessentially consists of (i) sodium chloride in a concentration of about150 mM and (ii) dextran of a molecular weight of about 70,000 Dalton inan amount of about 3% (w/w).

In a fifth aspect the invention provides a method of screening one ormore individuals for risk or future occurrence of a condition associatedwith hypertension. The method includes suspending erythrocytes of eachof the one or more individuals in a first sodium chloride solution ofabout physiological osmolality. The method also includes suspendingerythrocytes of each of the one or more individuals in a second sodiumchloride solution of about physiological osmolality. The first sodiumchloride solution has a sodium chloride concentration, which has a valuethat is about 25 mM or more below the value of the sodium chlorideconcentration of the second sodium chloride solution. The method furtherincludes allowing the erythrocytes suspended in the first sodiumchloride solution and the erythrocytes suspended in the second sodiumchloride solution to settle over a period of time. The suspendederythrocytes are allowed to settle for a period of time that issufficient to allow the formation of a supernatant. The method alsoincludes detecting the difference in height of the supernatant betweenthe first and in the second sodium chloride solution. As indicatedabove, the difference in height of the supernatant can generally also beassessed by detecting the difference in height of the settlederythrocytes, i.e. the height of the suspension that still containserythrocytes, between the first and in the second sodium chloridesolution. An increased difference in height of the supernatant betweenthe first and the second sodium chloride solution, relative to athreshold value, indicates an increased risk of future occurrence of acondition associated with hypertension.

In some embodiments the method according to the fifth aspect includescomparing the difference in height of the supernatant between the firstand the second sodium chloride solution to a control measurement or to apredetermined reference value. In some embodiments the threshold valueis based on the difference in height of the supernatant between acorresponding first and a corresponding second sodium chloride solutionof erythrocytes of a control sample.

If an increased difference in height of the supernatant between thefirst and the second sodium chloride solution has been detected, amethod according to the fifth aspect may include stratifying the subjectfor monitoring blood pressure.

In some embodiments the method of the fifth aspect further includesrepeatedly detecting the difference in height of the settlederythrocytes.

In some embodiments of the method according to the fifth aspect thefirst and the second sodium chloride solution include a polysaccharide,for example in an amount of about 3% (w/w).

If the polysaccharide is dextran, it typically has an average molecularweight of about 70,000 Dalton. Where a polysaccharide different fromdextran is included in the first and the second sodium chloridesolution, it may also have an average molecular weight of about 70,000Dalton. In some embodiments the polysaccharide has a molecular weight ofabout 70,000 Dalton.

In some embodiments one or both of the first and the second sodiumchloride solution include a monosaccharide or a disaccharide, such assucrose.

In some embodiments of the method according to the fifth aspect thefirst sodium chloride solution has a sodium chloride concentration ofabout 125 mM, includes about 3% (w/w) dextran of a molecular weight ofabout 70,000 Dalton and 50 mM sucrose. In some of these embodiments thesecond sodium chloride solution has a sodium chloride concentration ofabout 150 mM and includes about 3% (w/w) dextran of a molecular weightof about 70,000 Dalton. In one embodiment the first sodium chloridesolution is an aqueous solution that at least essentially consists of(i) sodium chloride in a concentration of about 125 mM, (ii) dextran ofa molecular weight of about 70,000 Dalton in an amount of about 3% (w/w)and (iii) sucrose in a concentration of about 50 mM. In this embodimentthe second sodium chloride solution is an aqueous solution that at leastessentially consists of (i) sodium chloride in a concentration of about150 mM and (ii) dextran of a molecular weight of about 70,000 Dalton inan amount of about 3% (w/w).

In a sixth aspect there is provided a method of monitoring the risk ofoccurrence of hypertension in a subject. The method includes monitoringthe difference in height of the supernatant between a first and a secondsuspension of erythrocytes of the subject in a sodium chloride solutionof about physiological osmolality after having allowed the suspendederythrocytes to settle for a period of time. The first sodium chloridesolution has a sodium chloride concentration that is at least about 25mM lower than the sodium chloride concentration of the second sodiumchloride solution. An increased difference in height of the supernatantbetween the first and the second sodium chloride solution, relative to athreshold value, indicates an increased risk of hypertension.

According to a particular embodiment of the method according to thesixth aspect, the difference in height of the supernatant is monitoredat certain, e.g. predetermined, time intervals.

In a seventh aspect the present invention provides a method of assessingthe risk of occurrence of a hypertension associated condition in asubject. The method includes detecting at least one of (i) the thickness(height) or the volume of the glycocalyx of erythrocytes of the subject,and (ii) the zeta potential of the erythrocytes, i.e. the degree ofelectrostatic repulsion between erythrocytes of the subject. If a heightor volume of the glycocalyx of the erythrocytes is detected that islower than a threshold value, the method includes monitoring thesubject's blood pressure. If a height or volume of the glycocalyx of theerythrocytes is detected that is about at or above a threshold value thesubject's blood pressure generally need not be monitored. If a zetapotential of the erythrocytes is detected that is lower than a thresholdvalue, the method includes monitoring the subject's blood pressure. If azeta potential of the erythrocytes is detected that is about at or abovea threshold value the subject's blood pressure generally need not bemonitored. In the method an increased risk of occurrence of ahypertension associated condition is diagnosed if an increased bloodpressure relative to a threshold value is detected.

In typical embodiments the method according to the seventh aspect alsoincludes comparing the blood pressure to a control measurement or to apredetermined reference value. An increase in systolic and/or diastolicblood pressure compared to the control measurement indicates that thesubject is at an increased risk of occurrence of a hypertensionassociated condition.

The hypertension associated condition is a condition that may occur as aresult of hypertension in the organism of a subject. In some embodimentsthe hypertension associated condition is arteriosclerosis, cardiacand/or kidney insufficiency, brain bleeding (stroke) or myocardialinfarction.

In some embodiments of the method according to the seventh aspect theinorganic salt is a sodium salt such as sodium chloride. In someembodiments a respective sodium salt is Na₂SO₄. In some embodiments arespective sodium salt is Na₂NO₃. In some embodiments the sodium salt isNa₂CO₃. In some embodiments the sodium salt is Na₂HPO₄ NaH₂PO₄ orNa₃PO₄. In some embodiments the inorganic salt is a potassium salt.Examples of a suitable potassium salt include, but are not limited to,KCl, Ka₂NO₃ and K₂SO₄. In some embodiments the inorganic salt is acalcium salt such as CaCl₃, Ca₃(PO₃)₂, Ca(NO₃)₂, CaCO₃ or CaCl₂. In someembodiments the inorganic salt is an aluminium salt, such as AlCl₃ orAl₂(SO₄)₃. In some embodiments the inorganic salt is a magnesium saltsuch as MgCl₂ or MgSO₄. In some embodiments the inorganic salt is alithium salt. A suitable lithium salt may for example be LiCl or Li₂SO₄.In some embodiments the inorganic salt is a zinc salt such as ZnSO₄,ZnCl₂ or Zn(SO₄)₂. In some embodiments the inorganic salt is a mixtureof at least two of a sodium salt, a potassium salt, a calcium salt, analuminium salt, a lithium salt, a magnesium salt, an iron salt, a coppersalt, a nickel salt and a zinc salt.

In some embodiments of the method according to the seventh aspect theheight and/or the volume of the glycocalyx of erythrocytes is analysedby atomic force microscopy.

In some embodiments of the method according to the seventh aspect thezeta potential of the erythrocytes is determined using a zeta potentialanalyser. In some embodiments the zeta potential is analysed bycomparing the aggregation tendency of erythrocytes in two differentsolutions, which both have an about physiological osmolality, but differin the concentration of the inorganic salt. In such an embodiment amethod according to the first aspect includes suspending erythrocytes ofthe subject in a first solution of an inorganic salt. The first solutionis of about physiological osmolality. The method of such an embodimentalso includes suspending erythrocytes of the subject in a secondsolution of an inorganic salt. The second solution is of aboutphysiological osmolality. In some embodiments the first and the secondsolution are both solutions of sodium chloride, with the first sodiumchloride solution having a sodium chloride concentration that is lowerthan the sodium chloride concentration of the second sodium chloridesolution. In one embodiment the sodium chloride concentration in thefirst sodium chloride solution is about 25 mM or more lower than thesodium chloride concentration of the second sodium chloride solution. Insome embodiments comparing the aggregation tendency of erythrocytes intwo different solutions of an inorganic salt includes allowing theerythrocytes suspended in the two solutions of an inorganic salt tosettle over a period of time. In one embodiment where the inorganic saltis sodium chloride the method includes allowing the erythrocytessuspended in the first sodium chloride solution and the erythrocytessuspended in the second sodium chloride solution to settle over a periodof time. The suspended erythrocytes are allowed to settle for a periodof time that is sufficient to allow the formation of a supernatant. Inembodiments where two different solutions of an inorganic salt areemployed for analysing the aggregation tendency of erythrocytes themethod further includes detecting the difference in height of thesupernatant between the two solutions of an inorganic salt. Inembodiments where the inorganic salt is sodium chloride the methodgenerally includes detecting the difference in height of the supernatantbetween the first and the second sodium chloride solution. Generally thedifference in height of the supernatant can also be detected bydetection the difference in height of the settled erythrocytes, i.e. theheight of the suspension that still contains erythrocytes, between thetwo solutions of the inorganic salt, such as the first and in the secondsodium chloride solution. An increased difference in height of thesupernatant between the two solutions of the inorganic salt, e.g.between the first and the second sodium chloride solution, relative to athreshold value, indicates that the blood pressure of the subject issensitive to sodium intake.

In an eighth aspect the present invention provides a method of assessingthe risk of occurrence of a hypertension associated condition in asubject. The method includes detecting the difference in height of thesupernatant between a first and a second suspension of erythrocytes ofthe subject in a sodium chloride solution of about physiologicalosmolality after having allowed the suspended erythrocytes to settle fora period of time. The period of time is sufficient to allow theformation of a respective supernatant. The sodium chloride solution ofthe first suspension of erythrocytes has a sodium chloride concentrationthat is at least about 25 mM lower than the sodium chloride solution ofthe second suspension of erythrocytes. Furthermore, if a difference inheight of the supernatants between the first and in the secondsuspension is detected that is increased relative to a threshold value,the method includes monitoring the subject's blood pressure. In themethod an increased risk of occurrence of a hypertension associatedcondition is diagnosed if an increased blood pressure relative to athreshold value is detected.

In typical embodiments the method according to the eighth aspect alsoincludes comparing the blood pressure to a control measurement or to apredetermined reference value.

An increase in systolic and/or diastolic blood pressure compared to thecontrol measurement indicates that the subject is at an increased riskof occurrence of a hypertension associated condition.

The hypertension associated condition is a condition that may occur as aresult of hypertension in the organism of a subject. In some embodimentsthe hypertension associated condition is arteriosclerosis, cardiacand/or kidney insufficiency, brain bleeding (stroke) or myocardialinfarction.

In a ninth aspect the present invention provides an in vitro method ofanalysing whether the blood pressure of a subject is sensitive to sodiumintake. The method includes suspending erythrocytes of the subject in asodium chloride solution of about physiological osmolality. The methodalso includes suspending erythrocytes of the subject in a solution thatcontains potassium chloride, which is at least essentially void ofsodium chloride—herein also referred to as a potassium chloridesolution. The potassium chloride solution is of about physiologicalosmolality. The concentrations of sodium chloride and potassium chloridein the sodium chloride solution and the potassium chloride solution,respectively, are of at least essentially the same value. The methodfurther includes allowing the erythrocytes suspended in the sodiumchloride solution and the erythrocytes suspended in the potassiumchloride solution to settle over a period of time. The suspendederythrocytes are allowed to settle for a period of time that issufficient to allow the formation of a supernatant. The method alsoincludes detecting the difference in height of the supernatant betweenthe sodium chloride solution and the potassium chloride solution.Generally the difference in height of the supernatant can also bedetected by detection the difference in height of the settlederythrocytes, i.e. the height of the suspension that still containserythrocytes, between the first and in the second sodium chloridesolution. An increased difference in height of the supernatant betweenthe sodium chloride solution and the potassium chloride solution,relative to a threshold value, indicates that the blood pressure of thesubject is sensitive to sodium intake.

In some embodiments the method according to the ninth aspect includescomparing the difference in height of the supernatant between the sodiumchloride solution and the potassium chloride solution to a controlmeasurement or to a predetermined reference value.

In some embodiments the threshold value is based on the difference inheight of the supernatant between a corresponding sodium chloridesolution and a corresponding potassium chloride solution of erythrocytesof a control sample.

If an increased difference in height of the supernatant between thesodium chloride solution and the potassium chloride solution has beendetected, a method according to the ninth aspect may include stratifyingthe subject for monitoring blood pressure.

In some embodiments of the method according to the ninth aspect repeatedsessions of the method according to the ninth aspect are carried out.Sessions of the method may be carried out at certain, e.g.predetermined, time intervals.

In some embodiments of the method according to the ninth aspect thesodium chloride solution and the potassium chloride solution include apolysaccharide, for example in an amount of about 3% (w/w). If thepolysaccharide is dextran, it typically has an average molecular weightof about 70,000 Dalton. Where another polysaccharide is included in thefirst and the second sodium chloride solution, it may also have anaverage molecular weight of about 70,000 Dalton. In some embodiments thepolysaccharide has a molecular weight of about 70,000 Dalton.

In some embodiments one or both of the sodium chloride solution and thepotassium chloride solution include a monosaccharide or a disaccharide,such as sucrose.

In some embodiments the sodium chloride solution has a sodium chlorideconcentration of about 125 mM, includes about 3% (w/w) dextran of amolecular weight of about 70,000 Dalton and 50 mM sucrose. In some ofthese embodiments the potassium chloride solution has a potassiumchloride concentration of about 150 mM and includes about 3% (w/w)dextran of a molecular weight of about 70,000 Dalton. In one embodimentthe sodium chloride solution is an aqueous solution that at leastessentially consists of (i) sodium chloride in a concentration of about125 mM, (ii) dextran of a molecular weight of about 70,000 Dalton in anamount of about 3% (w/w) and (iii) sucrose in a concentration of about50 mM. In this embodiment the potassium chloride solution may be anaqueous solution that at least essentially consists of (i) potassiumchloride in a concentration of about 125 mM, (ii) dextran of a molecularweight of about 70,000 Dalton in an amount of about 3% (w/w) and (iii)50 mM sucrose.

In some embodiments of the method according to the ninth aspect theperiod of time for allowing the suspended erythrocytes to settle isselected in the range from about 45 to about 120 minutes.

In some embodiments the method according to the ninth aspect includessuspending erythrocytes of the subject in a first pair of solutions asdescribed above. The first pair of solutions includes a first solution,being a sodium chloride solution of about physiological osmolality. Thefirst pair of solutions further includes a second solution, being apotassium chloride solution of about physiological osmolality. Themethod also includes suspending erythrocytes of the subject in a secondpair of solutions. The second pair of solutions includes a firstsolution, being a sodium chloride solution of about physiologicalosmolality. The second pair of solutions also includes a secondsolution, being a potassium chloride solution of about physiologicalosmolality. In one embodiment the concentration of sodium chloride inthe first solution of the first pair of solutions is at leastessentially the same as the concentration of potassium chloride in thesecond solution of the second pair of solutions.

In a tenth aspect the present invention provides an in vitro method ofassessing the risk of occurrence of hypertension in a subject. Themethod includes suspending erythrocytes of the subject in a sodiumchloride solution of about physiological osmolality. The method alsoincludes suspending erythrocytes of the subject in a potassium chloridesolution of about physiological osmolality. The concentrations of sodiumchloride and potassium chloride in the sodium chloride solution and thepotassium chloride solution are of at least essentially the same value.The method further includes allowing the erythrocytes suspended in thesodium chloride solution and the erythrocytes suspended in the potassiumchloride solution to settle over a period of time. The suspendederythrocytes are allowed to settle for a period of time that issufficient to allow the formation of a supernatant. The method alsoincludes detecting the difference in height of the supernatant betweenthe sodium chloride solution and the potassium chloride solution.Generally the difference in height of the supernatant can also bedetected by detection the difference in height of the settlederythrocytes, i.e. the height of the suspension that still containserythrocytes, between the sodium chloride solution and the potassiumchloride solution. An increased height of the supernatant relative to athreshold value, indicates an increased risk of occurrence ofhypertension.

In some embodiments the method according to the tenth aspect is a methodof diagnosing and/or stratifying the risk of developing hypertension. Insome embodiments the method according to the tenth aspect is a method ofassessing the risk of occurrence of a hypertension associated conditionin a subject. The hypertension associated condition may for instance beat least one of cardiovascular disease, kidney disease, hypertensiveretinopathy, dementia, and aortic dissection.

In some embodiments the method according to the tenth aspect includescomparing the difference in height of the supernatant between the sodiumchloride solution and the potassium chloride solution to a controlmeasurement or to a predetermined reference value.

In some embodiments the threshold value is based on the difference inheight of the supernatant between a corresponding sodium chloridesolution and a corresponding potassium chloride solution of erythrocytesof a control sample.

If an increased difference in height of the supernatant between thesodium chloride solution and the potassium chloride solution has beendetected, a method according to the tenth aspect may include stratifyingthe subject for monitoring blood pressure.

In some embodiments the method of the tenth aspect further includesrepeatedly detecting the difference in height of the settlederythrocytes.

In some embodiments of the method according to the tenth aspect thesodium chloride solution and the potassium chloride solution include apolysaccharide, for example in an amount of about 3% (w/w). If thepolysaccharide is dextran, it typically has an average molecular weightof about 70,000 Dalton. Where a polysaccharide different from dextran isincluded in the sodium chloride solution and the potassium chloridesolution, it may also have an average molecular weight of about 70,000Dalton. In some embodiments the polysaccharide has a molecular weight ofabout 70,000 Dalton.

In some embodiments one or both of the sodium chloride solution and thepotassium chloride solution include a monosaccharide or a disaccharide,such as sucrose.

In some embodiments of the method according to the tenth aspect thesodium chloride solution has a sodium chloride concentration of about150 mM and includes about 3% (w/w) dextran of a molecular weight ofabout 70,000 Dalton. In some of these embodiments the potassium chloridesolution has a potassium chloride concentration of about 150 mM andincludes about 3% (w/w) dextran of a molecular weight of about 70,000Dalton. In one embodiment the sodium chloride solution is an aqueoussolution that at least essentially consists of (i) sodium chloride in aconcentration of about 150 mM and (ii) dextran of a molecular weight ofabout 70,000 Dalton in an amount of about 3% (w/w). In such anembodiment the potassium chloride solution may be an aqueous solutionthat at least essentially consists of (i) potassium chloride in aconcentration of about 150 mM and (ii) dextran of a molecular weight ofabout 70,000 Dalton in an amount of about 3% (w/w).

In an eleventh aspect the present invention provides an in vitro methodof analysing whether the blood pressure of a subject is sensitive tosodium intake and/or whether the risk of occurrence of hypertension in asubject is increased. The method includes suspending erythrocytes of thesubject in in one or more pairs of a first solution and a secondsolution. Both the first solution and the second solution of each pairof a first solution and a second solution are of about physiologicalosmolality. Each first solution and each second solution contain atleast essentially only one of sodium chloride and potassium chloride.For each pair of first and second solution, the first solution containssodium chloride. For each pair of first and second solution, the secondsolution contains sodium chloride or potassium chloride. If a secondsolution of a pair of solutions contains sodium chloride, it has asodium chloride concentration that is at least about 25 mM higher thanthe sodium chloride concentration of the first solution. If a secondsolution of a pair of solutions contains potassium chloride, it has aconcentration of potassium chloride that is of at least essentially thesame value as the concentration of sodium chloride in the firstsolution. The method also includes allowing the suspended erythrocytesin the first and in the second solution of each pair of solutions tosettle for a period of time sufficient to allow the formation of asupernatant. Furthermore the method includes detecting the difference inheight of the supernatant between the first and the second solution ofeach pair of solutions. An increased difference in height of thesupernatants, relative to a threshold value, indicates that the bloodpressure of the subject is sensitive to sodium intake and/or that the isat an increased risk of occurrence of hypertension.

In some embodiments the method according to the eleventh aspect is amethod of assessing the risk of occurrence of a hypertension associatedcondition in a subject. The hypertension associated condition may forinstance be one or more of cardiovascular disease, kidney disease,hypertensive retinopathy, dementia, and aortic dissection.

In some embodiments the method according to the eleventh aspect includescomparing the difference in height of the supernatant between the sodiumchloride solution and the potassium chloride solution to a controlmeasurement or to a predetermined reference value. In some embodimentsthe threshold value is based on the difference in height of thesupernatant between a corresponding sodium chloride solution and acorresponding potassium chloride solution of erythrocytes of a controlsample.

In some embodiments of the method according to the eleventh aspect eachof the first and the second solution of a pair of a first solution and asecond solution has a concentration of sodium chloride or potassiumchloride that is about 100 mM or more.

If an increased difference in height of the supernatant between thesodium chloride solution and the potassium chloride solution has beendetected, a method according to the eleventh aspect may includestratifying the subject for monitoring blood pressure.

In some embodiments the method of the eleventh aspect further includesrepeatedly detecting the difference in height of the settlederythrocytes.

In some embodiments of the method according to the eleventh aspect thefirst and the second solution of a pair of a first solution and a secondsolution further include a polysaccharide, for example in an amount ofabout 3% (w/w). If the polysaccharide is dextran, it typically has anaverage molecular weight of about 70,000 Dalton. Where a polysaccharidedifferent from dextran is included in the first and the second solution,it may also have an average molecular weight of about 70,000 Dalton. Insome embodiments the polysaccharide has a molecular weight of about70,000 Dalton.

In some embodiments one or both of the first and the second solution ofa pair of a first solution and a second solution include amonosaccharide or a disaccharide, such as sucrose.

In some embodiments of the method according to the eleventh aspect thesecond solution has a sodium chloride concentration of about 150 mM andincludes about 3% (w/w) dextran of a molecular weight of about 70,000Dalton. In some embodiments of the method according to the eleventhaspect the first solution has a sodium chloride concentration of about100 mM, includes about 3% (w/w) dextran of a molecular weight of about70,000 Dalton and 100 mM sucrose. In some of these embodiments thesecond solution has a potassium chloride concentration of about 100 mM,includes about 3% (w/w) dextran of a molecular weight of about 70,000Dalton and 100 mM sucrose. In one embodiment the second solution is anaqueous solution that at least essentially consists of (i) sodiumchloride in a concentration of about 150 mM and (ii) dextran of amolecular weight of about 70,000 Dalton in an amount of about 3% (w/w).In such an embodiment the second solution may be an aqueous solutionthat at least essentially consists of (i) potassium chloride in aconcentration of about 100 mM, (ii) dextran of a molecular weight ofabout 70,000 Dalton in an amount of about 3% (w/w) and (iii) 100 mMsucrose.

In some embodiments of the method according to the eleventh aspect thethreshold value is based on the difference in height of the supernatantbetween a corresponding first and a corresponding second solution oferythrocytes of a control sample. In some embodiments of the methodaccording to the eleventh aspect the erythrocytes of the subject havebeen obtained by sedimentation of cells of blood from the subject,followed by removal of the buffy coat. In some embodiments of the methodaccording to the eleventh aspect suspending erythrocytes in the firstsodium chloride solution and in the second sodium chloride solutionfurther includes introducing the erythrocytes into a tube. A respectivetube may in some embodiments be a capillary, which has an open end and asealed end.

In some embodiments of the method according to the eleventh aspect theperiod of time for allowing the suspended erythrocytes to settle isselected in the range from about 45 to about 120 minutes.

In some embodiments the method according to the eleventh aspect includessuspending erythrocytes of the subject in a first and a second pair ofsolutions. Each pair of solutions encompasses a first solution and asecond solution. For the first pair of solutions, both the firstsolution and the second solution contain sodium chloride. For the secondpair of solutions the first solution contains sodium chloride, and thesecond solution contains potassium chloride. The concentration of sodiumchloride in the first solution of the first pair of solutions is atleast essentially the same as the concentration of potassium chloride inthe second solution of the second pair of solutions.

In a twelfth aspect the present invention provides an in vitro method ofanalysing whether the blood pressure of a subject is sensitive to sodiumintake and/or whether the risk of occurrence of hypertension in asubject is increased. The method includes suspending erythrocytes of thesubject in a first solution and a second solution. Both the firstsolution and the second solution are of about physiological osmolality.The first solution and the second solution contain at least essentiallyonly one of sodium chloride and potassium chloride. The first solutioncontains sodium chloride and the second solution contains potassiumchloride. The second solution has a potassium chloride concentrationthat is at least about 25 mM higher than the sodium chlorideconcentration of the first solution. The method also includes allowingthe suspended erythrocytes in the first and in the second solution tosettle for a period of time sufficient to allow the formation of asupernatant. Furthermore the method includes detecting the difference inheight of the supernatant between the first and the second solution. Anincreased difference in height of the supernatants, relative to athreshold value, indicates that the blood pressure of the subject issensitive to sodium intake and/or that the is at an increased risk ofoccurrence of hypertension.

In some embodiments the method according to the twelfth aspect is amethod of assessing the risk of occurrence of a hypertension associatedcondition in a subject. The hypertension associated condition may forinstance be one or more of cardiovascular disease, kidney disease,hypertensive retinopathy, dementia, and aortic dissection.

In some embodiments the method according to the twelfth aspect includescomparing the difference in height of the supernatant between the sodiumchloride solution and the potassium chloride solution to a controlmeasurement or to a predetermined reference value. In some embodimentsthe threshold value is based on the difference in height of thesupernatant between a corresponding sodium chloride solution and acorresponding potassium chloride solution of erythrocytes of a controlsample.

In some embodiments of the method according to the twelfth aspect thefirst and the second solution has a concentration of sodium chloride orpotassium chloride that is about 100 mM or more.

If an increased difference in height of the supernatant between thesodium chloride solution and the potassium chloride solution has beendetected, a method according to the twelfth aspect may includestratifying the subject for monitoring blood pressure.

In some embodiments the method of the twelfth aspect further includesrepeatedly detecting the difference in height of the settlederythrocytes.

In some embodiments of the method according to the twelfth aspect thefirst and the second solution further include a polysaccharide, forexample in an amount of about 3% (w/w).

If the polysaccharide is dextran, it typically has an average molecularweight of about 70,000 Dalton. Where a polysaccharide different fromdextran is included in the first and the second solution, it may alsohave an average molecular weight of about 70,000 Dalton. In someembodiments the polysaccharide has a molecular weight of about 70,000Dalton.

In some embodiments one or both the first and the second solutioninclude a monosaccharide or a disaccharide, such as sucrose.

In some embodiments of the method according to the twelfth aspect thesecond solution has a potassium chloride concentration of about 125 mM,includes about 3% (w/w) dextran of a molecular weight of about 70,000Dalton and 50 mM sucrose. In some embodiments of the method according tothe twelfth aspect the first solution has a sodium chlorideconcentration of about 100 mM, includes about 3% (w/w) dextran of amolecular weight of about 70,000 Dalton and 100 mM sucrose. In such anembodiment the second solution may be an aqueous solution that at leastessentially consists of, or consists of, (i) potassium chloride in aconcentration of about 150 mM and (ii) dextran of a molecular weight ofabout 70,000 Dalton in an amount of about 3% (w/w).

In some embodiments of the method according to the twelfth aspect thethreshold value is based on the difference in height of the supernatantbetween a corresponding first and a corresponding second solution oferythrocytes of a control sample. In some embodiments of the methodaccording to the twelfth aspect the erythrocytes of the subject havebeen obtained by sedimentation of cells of blood from the subject,followed by removal of the buffy coat. In some embodiments of the methodaccording to the twelfth aspect suspending erythrocytes in the first andin the second solution further includes introducing the erythrocytesinto a tube. A respective tube may in some embodiments be a capillary,which has an open end and a sealed end.

In some embodiments of the method according to the twelfth aspect theperiod of time for allowing the suspended erythrocytes to settle isselected in the range from about 45 to about 120 minutes.

In a thirteenth aspect the invention provides a kit of parts. The kitincludes a first and a second container as well as a pair of tubes. Thefirst container includes a sodium chloride solution. The secondcontainer includes a sodium chloride solution, which has a sodiumchloride concentration that is higher than the sodium chlorideconcentration of the sodium chloride solution in the first container.

In some embodiments the sodium chloride solution in the second containerof the kit has a sodium chloride concentration that is at least about 25mM higher than the sodium chloride concentration in the first container.In some embodiments the sodium chloride solution in each of the firstand the second container is of about physiological osmolality. Thesodium chloride solution in each of the first and the second containerhas in some embodiments a sodium chloride concentration of about 100 mMor more.

In some embodiments the first and the second container further include amonosaccharide such as for instance glucose.

In some embodiments of the kit according to the thirteenth aspect thesodium chloride solutions in the first and in the second containerinclude a polysaccharide, for example in an amount of about 3% (w/w). Ifthe polysaccharide is dextran, it typically has an average molecularweight of about 70,000 Dalton. Where a polysaccharide different fromdextran is included in the sodium chloride solutions, it may also havean average molecular weight of about 70,000 Dalton. In some embodimentsthe polysaccharide has a molecular weight of about 70,000 Dalton.

In some embodiments of the kit one or both of the first and the secondsodium chloride solution include a monosaccharide or a disaccharide,such as sucrose.

In some embodiments the sodium chloride solution in the first containerof the kit has a sodium chloride concentration of about 125 mM, includesabout 3% (w/w) dextran of a molecular weight of about 70,000 Dalton and50 mM sucrose. In some of these embodiments the the sodium chloridesolution in the second container of the kit has a sodium chlorideconcentration of about 150 mM and includes about 3% (w/w) dextran of amolecular weight of about 70,000 Dalton. In one embodiment the sodiumchloride solution in the first container is an aqueous solution that atleast essentially consists of (i) sodium chloride in a concentration ofabout 125 mM, (ii) dextran of a molecular weight of about 70,000 Daltonin an amount of about 3% (w/w) and (iii) sucrose in a concentration ofabout 50 mM. In this embodiment the sodium chloride solution in thesecond container is an aqueous solution that at least essentiallyconsists of (i) sodium chloride in a concentration of about 150 mM and(ii) dextran of a molecular weight of about 70,000 Dalton in an amountof about 3% (w/w).

In a fourteenth aspect the invention provides a kit of parts. The kitincludes one or more pairs of a first and a second container, as well asa pair of tubes. A first container includes a first solution and asecond container includes a second solution. Each first solution andeach second solution contain at least essentially only one of sodiumchloride and potassium chloride. For each pair of a first and a secondcontainer, the first solution includes sodium chloride, and the secondsolution includes sodium chloride or potassium chloride. A secondsolution that contains potassium chloride has a concentration ofpotassium chloride that is of at least essentially the same value as theconcentration of sodium chloride in the first solution.

The second container includes a potassium chloride solution, which has apotassium chloride concentration that is of at least essentially thesame value as the sodium chloride concentration of the sodium chloridesolution in the first container.

In some embodiments the first and the second solution of one pair of afirst and a second container are of about physiological osmolality. Insome embodiments the first and the second solution of each pair of afirst and a second container are of about physiological osmolality. Thefirst and the second solution have in some embodiments a concentrationof sodium chloride and of potassium chloride, respectively, of about 100mM or more.

In some embodiments of the kit according to the fourteenth aspect, forone or more pairs of a first and a second container, a second solutioncontaining sodium chloride has a sodium chloride concentration that isat least about 25 mM higher than the sodium chloride concentration ofthe first solution.

In some embodiments the first and the second container further include amonosaccharide such as for instance glucose. In some embodiments thefirst and the second container include a disaccharide such as sucrose.

In some embodiments of the kit according to the fourteenth aspect, inone or more pairs of a first and a second container the first and thesecond solution further contain a polysaccharide. In some embodimentseach of the first and the second solution further contain apolysaccharide, i.e. for each pair of a first and a second container.

In some embodiments the first and the second solution of one or morepairs of a first and a second container include a polysaccharide, forexample in an amount of about 3% (w/w). If the polysaccharide isdextran, it typically has an average molecular weight of about 70,000Dalton. Where a polysaccharide different from dextran is included in thesodium chloride solutions, it may also have an average molecular weightof about 70,000 Dalton. In some embodiments the polysaccharide has amolecular weight of about 70,000 Dalton.

In some embodiments of the kit according to the thirteenth aspect eachof the tubes of the pair of tubes is a capillary, the capillary havingan open end and a sealable end.

In a fourteenth aspect the invention relates to the in-vitro use of akit of parts according to the twelfth aspect or according to thethirteenth aspect for assessing the risk of occurrence of hypertensionin a subject or for assessing the risk of occurrence of a hypertensionassociated condition in a subject.

In a fifteenth aspect the invention relates to the in-vitro use of a kitof parts according to the twelfth aspect or according to the thirteenthfor assessing the risk of occurrence of hypertension in a subject or forassessing the risk of occurrence of a hypertension associated conditionin a subject.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates, without being bound by theory, an assumption on thepossible basic principle underlying a method as disclosed herein. Anerythrocyte is carrying a negatively charged boundary layer, theglycocalyx, which attracts positive charges and repels negative charges.

FIG. 2 illustrates the glycocalyx present on red blood cells.

FIG. 3 depicts the sedimentation of aggregated erythrocytes over a timeinterval from 60 to 120 minutes. The figure can also serve as anillustration on determining erythrocyte salt sensitivity (ESS).

FIG. 4 depicts the sodium dependency of the negative zetapotential—detected by the height of the supernatant after allowingerythrocytes to settle.

FIG. 5 depicts the detection of results after allowing erythrocytes ofsamples from two volunteers to settle. Tubes depicted on the right ineach case contained 125 mM sodium (L₁), and tubes depicted on the leftcontained 150 mM sodium (L₂). At the bottom of each figure theindividual ESS value is calculated.

FIG. 6 depicts the erythrocyte salt sensitivity of 12 healthyindividuals. The grey bar in the center of the figure shows the mean ofall volunteers including the standard error. “Group A” consists of 3volunteers who are statistically significantly less sensitive to saltthan the average (p<0.01). “Group B” consists of 3 volunteers who arestatistically significantly more sensitive to salt than the average(p<0.01).

FIG. 7 depicts an image of erythrocytes obtained by atomic forcemicroscopy.

FIG. 8 schematically illustrates the visualisation of the glycocalyx oferythrocytes by differential imaging as shown in FIG. 9.

FIG. 9 depicts atomic force microscopy images of (A) erythrocytes beforethe removal of the glycocalyx and (B) erythrocytes after removal of theglycocalyx, as well as (C) a differential image (A-B) showing only theglycocalyx.

FIG. 10 depicts data (glycocalyx height) of monitoring by atomic forcemicroscopy the removal of the glycocalyx on erythrocytes by means ofheparinase.

FIG. 11 illustrates the progress of glycocalyx removal on erythrocytesdepicted by atomic force microscopy.

FIG. 12 depicts data (glycocalyx volume) of monitoring, by atomic forcemicroscopy, the removal of the glycocalyx on erythrocytes by means ofheparinase. Volume is indicated in femtoliters, i.e. units correspondingto 10⁻¹⁵ liters.

FIG. 13 depicts atomic force microscopy images of erythrocytes afterexposure to intact endothelium.

FIG. 14 depicts atomic force microscopy images of erythrocytes afterexposure to glycocalyx-depleted endothelium.

FIG. 15 shows the interrelation between intactness of endothelial anderythrocyte glycocalyx. Glycocalyx height of erythrocytes exposed tointact endothelium (black) is substantially greater than glycocalyxheight of erythrocytes exposed to glycocalyx-depleted endothelium(white). The value of glycocalyx-depleted erythrocytes exposed to intactendothelium is depicted as a white square.

FIG. 16 illustrates a comparison of the difference in height ofsupernatant after erythrocytes have been allowed to settle for 60minutes, which were suspended in a solution of 125 mM sodium chloride.The erythrocytes had been exposed (A) to intact endothelium and (B) toglycocalyx depleted endothelium.

FIG. 17 depicts the rate of sedimentation of erythrocytes which wereallowed to settle after exposure to intact endothelium (black bar) andto glycocalyx depleted endothelium (white bar).

FIG. 18 depicts the stiffness and the thickness (height) of theglycocalyx, as determined by atomic force microscopy, of blood vesselsafter 5 days of treatment with chronic low sodium (black dots), andtreatment with chronic high sodium (white dots). As can be taken fromthe figure, constant exposure to chronic high sodium levels causes theendothelial glycocalyx to shrink and stiffen.

FIG. 19 shows the erythrocyte sodium sensitivity (triple measurements)of 61 study participants. The value is 4.28±0.19 (n=61). Five studyparticipants explicitly indicated to be on a low-salt diet (white bars).

FIG. 20 depicts a comparison of two embodiments of a salt blood testaccording to the present disclosure. An embodiment using capillary blood(fingertip) was compared to an embodiment using venous puncture (venousblood) in 6 test persons. Paired study, i.e. venous blood and capillaryblood were taken from the same person and analysed on the same day. Thecorrelation of a linear regression line (order 1; not shown in graph) is0.91.

FIG. 21 illustrates a method disclosed herein, in which salt sensitivityis analysed in terms of sodium selectivity. Capillary blood from thefingertip was obtained from 12 study participants. The Na⁺ over K⁺selectivity i.e. the ratio of erythrocyte sedimentation rates in 125 Na⁺over 125 K⁺ solutions (ESSK=L125 Na⁺/L125 K⁺) are shown. A mean value of3.11±0.21 indicates that in average Na⁺ binds about 3-times moreselective as compared to K⁺.

FIG. 22 shows the frequency distribution of the erythrocyte sodiumsensitivity in a cohort of 61 study participants.

DETAILED DESCRIPTION OF THE BEST MODE OF THE INVENTION

Provided are methods of analysing the sensitivity of a subject's bloodpressure to sodium intake and of predicting the susceptibility of asubject to develop hypertension. Methods are described that can becarried out rapidly and easily. A method or use provided in thisspecification may in some embodiments be a method for risk assessment, amethod for diagnosis or prognosis of the occurrence of hypertensionand/or a hypertension related condition in a subject. In someembodiments a method or use described herein relates to the assessmentof prehypertension in a subject, i.e. a subject with a systolic bloodpressure of values in the range from about 120 to about 139 mm Hg and/ora diastolic blood pressure of values in the range from about 80 to about89 mm Hg.

A method or use described herein may be used in diagnosis or prognosisassessment of hypertension and/or a hypertension related condition in asubject, generally a mammalian subject. A respective use or method canprovide an indication of a risk of developing hypertension in a subject.The respective method or use may also be used to determine whether theblood pressure of a subject is sensitive to sodium intake. A positiveassessment in this regard will generally be an indication that thevascular system of the respective subject is sensitive to changes insodium intake. Put differently, a subject's blood pressure is sensitiveto sodium intake, if an intake of sodium, generally by ingestion of foodwith a high amount of sodium, can lead to an increase of the subject'sblood pressure. This term is generally to be understood as a relativeterm in that a subject's blood pressure is sensitive to sodium intake ifan intake of a given amount of sodium, typically per kg body weight,causes an increase of blood pressure that is higher than the increase ofblood pressure induced by the same amount of sodium in the organism ofan average individual with a comparable age. Further, a method or use asdisclosed herein may also be used in stratifying a subject forhypertension prevention measures or for hypertension therapy. An“individual” or “subject” as used herein refers to any mammal, includinge.g. a rabbit, a mouse, a Guinea pig, a hamster, a dog, a pig, a cow, asheep, a horse, a macaque, a gibbon and a human. A respective mammal mayin some embodiments be a veterinary animal such as a farm animal, adomestic animal or laboratory animal. Where the subject is a human, thesubject may be a patient.

The present invention is based on the surprising finding that thefunctional state of the glycocalyx of erythrocytes reflects thefunctional state of the endothelial glycocalyx. Therefore the functionalstate of both the glycocalyx of erythrocytes and the endothelialglycocalyx are useful prognostic parameters for a disposition ofhypertension. The term “hypertension” as used herein refers to higharterial blood pressure. Generally high arterial blood pressure ischaracterized by a systolic blood pressure that is consistently over 140mm Hg and/or a diastolic blood pressure that is consistently over 90 mmHg. If either or both of the systolic blood pressure and the diastolicblood pressure are too high, a subject has hypertension.

A subject to be diagnosed, stratified, screened or for whom/which anassessment is to be made is generally a subject that does not or not yetsuffer from a hypertension induced condition. Typically the subject doesnot have a history of a hypertension induced condition such as avascular disease, e.g. stroke or myocardial infarction. Typically thesubject does not suffer from atherothrombosis. In some embodiments thesubject is not suffering from an inflammatory condition of the vascularsystem. In some embodiments the subject does not (yet) suffer fromhypertension. In some embodiments the subject does not suffer from anacute disease or from atherothrombosis.

The erythrocytes used in a method or use according to this disclosurehave been obtained from the individual before carrying out themethod/use. The erythrocytes may in some have been kept at roomtemperature, i.e. about 18° C. The erythrocytes may also have been keptat 37° C. The erythrocytes may also have been kept at a temperature inthe range between 18° C. and 37° C. The erythrocytes used in a method oruse described herein may have been isolated from a blood sample obtainedthe individual before carrying out the method/use and subsequen havebeen kept at a temperature in the range from about 18° C. to about 37°C. Before isolating the erythrocytes the blood sample may likewise havebeen kept at 37° C. The sample may also have been kept at a temperaturein the range between 18° C. and 37° C. In some embodiments theerythrocytes isolated from the blood sample may have been kept at atemperature in the range from about 2° C. to about 37° C., such as fromabout 4° C. to about 37° C. or below. In some embodiments the theerythrocytes may have been kept at about 32° C. or below. In someembodiments the erythrocytes may have been kept at a temperature ofabout 25° C. or below. As an illustrative example, a whole blood samplemay be kept at about 25° C. or below.

The word “about” as used herein refers to a value being within anacceptable error range for the particular value as determined by one ofordinary skill in the art, which will depend in part on how the value ismeasured or determined, i.e., the limitations of the measurement system.For example, “about” can mean within 1 or more than 1 standarddeviation, per the practice in the art. The term “about” is also used toindicate that the amount or value in question may be the valuedesignated or some other value that is approximately the same. Thephrase is intended to convey that similar values promote equivalentresults or effects according to the invention. In this context “about”may refer to a range above and/or below of up to 10%. The word “about”refers in some embodiments to a range above and below a certain valuethat is up to 5%, such as up to up to 2%, up to 1%, or up to 0.5% aboveor below that value. In one embodiment “about” refers to a range up to0.1% above and below a given value.

As used herein, the conjunctive term “and/or” between multiple recitedelements is understood as encompassing both individual and combinedoptions. For instance, where two elements are conjoined by “and/or”, afirst option refers to the applicability of the first element withoutthe second. A second option refers to the applicability of the secondelement without the first. A third option refers to the applicability ofthe first and second elements together. Any one of these options isunderstood to fall within the meaning, and therefore satisfy therequirement of the term “and/or” as used herein. Concurrentapplicability of more than one of the options is also understood to fallwithin the meaning, and therefore satisfy the requirement of the term“and/or” as used herein.

In some embodiments the blood sample has been taken on the same day oron the previous day, such as about 48 hours or about 42 hours, beforethe method described herein is being carried out. In some embodimentsthe blood sample has been taken about 36 hours before carrying out amethoddescribed herein. In some embodiments the blood sample has beentaken about 30 hours before carrying out a respective method. In someembodiments the blood sample has been taken about 28 hours or about 24hours before the method is being carried out. In some embodiments theblood sample has been taken about 18 hours before carrying out a methodor use as described herein. In some embodiments the blood sample hasbeen taken about 15 hours before the method is being carried out. Theblood sample may also have been taken about 12 hours earlier. The bloodsample may in some embodiments have been taken about 6 hours or lessearlier, i.e. before carrying out a method of this disclosure. In someembodiments the blood sample has been taken about 2 hours or less beforecarrying out the method or use. In some embodiments the blood sample hasbeen obtained about 30 minutes or less before carrying out the method oruse.

A method according to this disclosure can be carried out on enriched orisolated erythrocytes of the subject. The term “subject” as used herein,also addressed as an individual, refers to a human or non-human animal,generally a mammal. A subject may be a mammalian species such as arabbit, a mouse, a rat, a Guinea pig, a hamster, a dog, a cat, a pig, acow, a goat, a sheep, a horse, a monkey, an ape or a human. Thus, themethods, uses and compositions described in this document are applicableto both human and veterinary disease. As explained in more detail below,the sample has been obtained from the subject. Further, while a subjectis typically a living organism, a method or use described in thisdocument may also be used in post-mortem analysis. Where the subject isa living human who is receiving medical care for a disease or condition,it is also addressed as a “patient”.

By the use of the term “enriched” is meant that the erythrocytesconstitute a significantly higher fraction (such as 2-5 fold) of allcells present in a sample of interest such as a cell suspension than inthe natural source from which the sample was obtained. The cell may alsoconstitute a significantly higher fraction than in an organism, whethernormal or diseased. This can most conveniently be achieved bypreferential reduction in the amount of other cells present, although apreferential increase in the amount of erythrocytes or a combination ofthe two may likewise be applied. However, it should be noted thatenriched does not imply that there are no other cells present. The termmerely defines that the relative amount of erythrocytes has beensignificantly increased. The term significant here is used to indicatethat the level of increase is useful to the person achieving such anincrease, and generally means an increase relative to other amino acidor nucleic acid sequences of about at least 2-fold, for example at leastabout 5- to 10-fold or even more. The term is meant to cover only thosesituations in which man has intervened to increase the proportion oferythrocytes.

The term “isolated” indicates that the cell or cells has/have beenremoved from its/their normal physiological environment, e.g. a naturalsource. An isolated cell or isolated cells may for instance be includedin a different medium such as an aqueous solution than providedoriginally, or placed in a different physiological environment.Typically isolated cells constitute a higher fraction of the total cellspresent in their environment, e.g. suspension, than in the environmentfrom which they were taken. The term “isolated” does not imply thaterythrocytes are the only cell type present, but that these cells areessentially free, e.g. about 80-90% pure or more, of other cells,respectively, in particular cells naturally associated with these cells.

Erythrocytes of the subject can conveniently be obtained from a bloodsample of the subject since the majority of cells in blood areerythrocytes. Thus centrifugation of blood, for instance at 500×g, willfor instance result in a cell pellet that consists mainly oferythrocytes, which can be recovered after removal of the supernatant.Most of the white blood cells and platelets are found in a so calledbuffy coat, a whitish sediment forming a layer above the erythrocytepellet. This buffy coat can thus likewise be removed. Where desired theobtained cell pellet may be washed once or several times by resuspendingin a suitable buffer, centrifugation and removal of supernatant. Anyother method of enriching or isolating red blood cells may likewise beemployed in order to obtain erythrocytes of a subject. An illustrativefurther example of obtaining erythrocytes is cell chromatography.

Over the past years clinical detection methods such as the method ofpulse wave analysis (Gurovich A N & Braith R W, Hypertens Res (2011) 34,166-169) or various imaging technologies (e.g. intima media thickness)have been refined to a degree that the structural/functional state ofthe vessel system can be conceived in detail. Apart from the relativecomplexity of these examinations, which does not permit a widespread usethroughout the population for purely preventive purposes, these modernmethods do only detect the state of the blood vessels once alreadyvisible (structural) changes have occurred. Therefore, these methods arenot suitable for the early diagnosis of a disposition for hypertension.Furthermore such elaborate methods are relatively complex since imagingof a subject is required.

As explained above, a method described herein makes use of the findingthat the functional state of the glycocalyx of erythrocytes can be usedas a prognostic parameter to identify a subject's disposition tohypertension. It has previously been suggested that electrostaticrepulsive force prevents aggregation of erythrocytes and thatNeuraminidase treatment increases the degree of aggregation oferythrocytes (Jan, K.-M., and Chien, S., J. Gen Physiol. (1973) 61,638-654). The present inventor verified this observation and developeddiagnostic methods that can be used as a quick test to inter aliaidentify subjects that are susceptible to develop hypertension as wellas susceptible to cardiovascular diseases, and to determine whether anindividual's blood pressure is different in sodium sensitivity, such asmore sensitive or less sensitive to sodium intake than an averageindividual of comparable health and age.

As is well known in the art, the inner wall of blood vessels is linedwith a layer of endothelial cells, which are of high importance for thefunction of blood vessels. On this endothelial surface there is aso-called endothelial glycocalyx (eGC), an anionic glycoprotein layer ofabout 500 nm in thickness and rich in water. The eGC participates in theregulation of vascular permeability, in the control of flow- andpressure-induced mechanotransduction of the endothelium and may play acrucial role in the pathogenesis of inflammation. Proteoheparan sulphatemacromolecules, anchored in the plasma membrane expose negativelycharged glycosaminoglycan side chains with binding sites for inorganiccations. This eGC is capable of temporarily buffering sodium of theblood. Recent research results support the view that the functionalstate of this eGC has a determining influence on the transport of sodiumfrom the blood stream into tissue (Oberleithner, H., et al., PflügersArch (2011) 462, 519-528). In other words, an intact eGC defines afunctional barrier for the resorption of sodium ions that have enteredthe blood. The ions can thus be eliminated via the kidneys and do nothave to take a route through the entire organism, involving a transientdeposit in tissues and organs (cf. FIG. 1). The eGC may play a prominentrole as a buffer barrier for sodium.

As illustrated in FIG. 1 and FIG. 2, erythrocytes likewise have aglycocalyx, which defines a soft surface of macromolecules surroundingan erythrocyte. As can be taken from FIG. 9C, FIG. 10 and FIG. 15, theglycocalyx thickness of red blood cells is much higher than the fewnanometers previously assumed, resulting in a contribution of theglycocalyx to the volume of erythrocytes that cannot be disregarded(FIG. 12). The negative surface charge of residues of the glycocalyxgenerates an electrostatic repulsive force. Without being bound by anyparticular theory it can thus be assumed that changes to the glycocalyxthickness will also modulate the charges per membrane surface area. Suchchanges will affect the net charge of an erythrocyte and thereby therepulsive force it exerts onto other erythrocytes. As erythrocytes forma cell suspension, a zeta potential can be detected, which characterizesthe degree of repulsion between cells, and thus the stability of thecorresponding cell suspension. The higher the zeta potential the lesslikely is aggregation to occur in the cells suspension. In a method asdescribed herein an indirect indication of the zeta potential is used,namely the rate of aggregation of erythrocytes. This rate of aggregationcan conveniently be assessed by allowing the erythrocytes to settle atdifferent salt concentrations.

In a method disclosed herein erythrocytes are being suspended in twosolutions. These two solutions may in some embodiments be two or moresodium chloride solutions. Both sodium chloride solutions are of aboutphysiological osmolarity so that the erythrocytes remain intact whilethe method is being carried out. In some embodiments these two solutionsmay include a sodium chloride solution and a potassium chloridesolution.

It is understood that in the context of a method or use described hereinboth osmolarity and osmolality may be detected in order to adjustsuitable conditions with regard to solution used to suspend erythrocytestherein. Suitable conditions in this context are conditions that avoidthe formation of osmotic pressure into or out of erythrocytes from thesubject. Where two salt solutions of different salt concentrations areused, the osmotic conditions to which the erythrocytes are exposed areadjusted to be at least essentially comparable, including at leastessentially identical. Osmolarity is expressed as the number of soluteparticles per unit of volume, e.g. liter, of solvent, whereas osmolalityis expressed as the number of solute particles per unit of mass, e.g.kilogram, of solvent, typically water. Osmolality can conveniently bemeasured using an osmometer. As the skilled person is well aware,osmolarity is thus affected by changes in water content, temperature andpressure, while osmolality is independent of temperature and pressure.

In embodiments where two sodium chloride solutions are being employed,the two sodium chloride solutions differ in their sodium chlorideconcentration by a value of about 25 mM or more. As an illustrativeexample the two solutions may differ in their sodium chlorideconcentration by about 40 mM or more. As a result of the difference insodium chloride concentration the number of electrolytes present in thetwo solutions differs and the repulsive forces between erythrocytes inthe two solutions likewise differ. Thus a difference in the rate ofaggregation exists between erythrocytes in the two solutions.Accordingly, if erythrocytes with approximately the same cell number permillilitre are allowed to settle for a limited period of time insolutions with different sodium chloride concentrations the erythrocytesin the two solutions settle at a different rate. The present inventorhas observed a correlation in that the less repulsive forces existbetween the erythrocytes the greater is the difference in aggregationtendency. Thus if erythrocytes with approximately the same cell numberper millilitre are allowed to settle for a limited period of time in twosolutions with different sodium chloride concentrations, theerythrocytes in the solutions with the higher sodium chlorideconcentration will settle at a higher rate than the erythrocytes in thesolutions with the lower sodium chloride concentration. In someembodiments the two sodium chloride solutions differ in their sodiumchloride concentration by about 50 mM or more. In some embodiments thetwo sodium chloride solutions differ in their sodium chlorideconcentration by about 70 mM or more.

Generally in a method disclosed herein any means to detect erythrocytesedimentation velocity may be employed. Typically erythrocytesedimentation velocity is measured in aqueous solution, which mayadvantageously be selected to be an isosmotic electrolyte solution.Where two aqueous solutions are being used, which differ in their sodiumsalt concentration by about 25 mM or more, sedimentation velocities inthese two solutions can be detected and compared. A ratio of theerythrocyte sedimentation velocity in a solution of a higher sodium saltconcentration over erythrocyte sedimentation velocity in a solution of alower sodium salt concentration can be determined, herein also calledthe erythrocyte sodium sensitivity (ESS). This erythrocyte sodiumsensitivity is inversely related to erythrocyte sodium buffer capacity(cf. above). In one embodiment the ratio of the erythrocytesedimentation velocities in a solution of a sodium salt such as sodiumchloride of 150 mM and 125 mM is being determined. In such an embodimentthe ESS is expressed as the ratio of the erythrocyte sedimentationvelocities of 150 mM over 125 mM Na⁺ solutions. the Na⁺ solutions.

In some embodiments a sodium chloride solution and a potassium chloridesolution are being employed. Without being bound by theory, thefollowing explanations may provide an illustration on how the methoddeveloped by the inventor can be envisaged to find a physiologicalbackground. Erythrocyte sedimentation rate in a defined electrolytesolution not only depends on ion strength but also on the ion species.There are difference in the properties of the specific ion, such as thesize of the ion, the charge density on its surface and binding capacityof water molecules to its surface, which may be envisaged to beinvolved. A hypothetic model on the effect of an ion's charge density inaqueous solution, its enthalpy of hydration, and the resulting effectson interactions in biological systems has been given by Collins(Biophysical Journal (1997) 72, 65-76).

Na⁺ is a so-called kosmotropic ion, also called antichaotropic, i.e. asmall cation with high electrical charge density at its surface. Becauseof this specific property a Na⁺ ion is thought to bind water moleculestightly (“water maker”). Due to the specific properties and the highconcentration of Na⁺ in extracellular fluids including human plasma thiscation is the major binding partner for the negatively chargedglycocalyx. The electrical surface charge properties of the erythrocyteglycocalyx (zeta potential) determine erythrocyte sedimentation rate indefined protein-free electrolyte solution. As described elsewhere hereinthe erythrocyte sodium sensitivity (ESS) can be determined by usingspecific electrolyte solutions of two different Na⁺ concentrations. ESSis an indirect measure of vascular sodium sensitivity. A large ESSindicates a poor Na⁺ buffering power of the glycocalyx and vice versa.From the erythrocyte sodium sensitivity a conclusion can be drawn on thesodium buffering capacity of the erythrocyte glycocalyx, and indirectlyon that of the endothelial glycocalyx.

An extention of this hypothesis is the comparison of Na⁺ and K⁺ bindingto the negatively charged glycocalyx. In contrast to kosmotropic sodium,K⁺ is classified as being chaotropic (Collins, K. D., 1997, supra). K⁺is thought to be larger in size compared to Na⁺, with a surface chargedensity that is smaller, and water binding being weak. These theoreticconclusions may explain, why it is unexpectedly possible to assess asubject's blood pressure sensitivity by comparing erythrocytesedimentation rates in two or more separate electrolyte solutions, eachof which essentially containing only one of Na⁺ and K⁺. One solutionused contains Na⁺, and one solution K⁺. The solutions containing Na⁺ andK⁺, respectively, have at least essentially the same ionic strength ofthe respective ion. In one embodiment the solutions containing Na⁺ andK⁺, respectively, have the same ionic strength of the respective ion. Bycomparing sedimentation rates in these solutions, an additionalcriterion of the binding properties of the glycocalyx can be obtained.It is herein termed ESSK, and it is defined by the ratio of thesedimentation rate in a solution containing a sodium salt to the rate ina solution containing a potassium salt. In typical embodiments the ESSKis the ratio of the sedimentation rate in a solution containing Na⁺ overthe sedimentation rate in a solution K⁺, where the concentration ofsodium and potassium are selected in the range from about 100 mM toabout 150 mM. In some embodiments the concentration of sodium andpotassium are selected in the range from about 110 mM to about 140 mM.The concentration of sodium and potassium may for example be about 115mM or about 135 mM. In one embodiment the ESSK is the ratio of thesedimentation rate in a solution containing 125 mM Na⁺ over thesedimentation rate in a solution containing 125 mM K.

Where for erythrocytes from a subject a large ratio of sedimentation ina sodium salt solution vs. potassium salt solution is determined, thisindicates high selectivity of specific Na⁺ binding, when compared to K⁺,to the negatively charged glycocalyx. If a low ratio of sedimentation ina sodium salt solution vs. potassium salt solution is determined, thisindicates low selectivity of specific Na⁺ binding to the glycocalyx,when compared to K⁺. Since chronic Na⁺ overload is thought to damage theendothelial glycocalyx (reflected by the respective erythrocyte zetapotential), an increased value of ESSK indicates a strong Na⁺ affinitywhen compared to K⁺, and thus a poor glycocalyx. Accordingly, if a largeratio of sedimentation in a sodium salt solution vs. potassium saltsolution is detected, it can be assessed that the respective subject issensitive to sodium intake and at an increased risk to develophypertension.

In some embodiments both the ESS and the ESSK are being assessed. Asexplained explained above, the ESS is the ratio of erythrocytesedimentation velocities in higher over lower sodium salt concentration,for instance 150 mM Na⁺ over 125 mM Na⁺. It can be taken to define ameasure for the Na⁺ sensitivity of the glycocalyx. The ESSK is the ratioof erythrocyte sedimentation velocities in sodium over potassium saltconcentration, for instance 125 mM Na⁺ over 125 mM K. It can be taken todefine a measure for the Na⁺ selectivity of the glycocalyx. A high ESSindicates a low Na⁺ binding capacity of the glycocalyx and vice versa. Ahigh ESSK indicates a high affinity for Na⁺ binding of the glycocalyxand vice versa. Low-capacity/high-affinity for Na⁺ can be taken tocharacterize a poor glycocalyx, a high-capacity/low-affinity tocharacterize a well-developed glycocalyx.

In embodiments where both the ESS and the ESSK are being assessed, as anoverall parameter for the quality/quantity of the glycocalyx both ratioscan be multiplied with each other. This term is herein also referred toas OSS (overall sodium sensitivity). If the above exemplaryconcentrations of 150 mM Na⁺ over 125 mM Na⁺, as well as 125 mM Na⁺ over125 mM K⁺ are used, the OSS derives from:

OSS=ESS×ESSK

OSS=(150 mM Na⁺/125 mM Na⁺)×(125 mM Na⁺/125 mM K⁺)

In this equation 125 mM Na⁺ can be canceled. Then, OSS consists only oftwo remaining determinants to be measured. It reads as follows:

OSS=150 mM Na⁺/125 mM K⁺

It is understood that instead of the exemplary 150 mM sodium and the 125mM potassium other values may be applicable, according to the solutionsselected. However, the above simplified equation only applies if thelower sodium concentration of the ratio of erythrocyte sedimentationvelocities in higher over lower sodium salt concentration equals theconcentration used for the ratio of erythrocyte sedimentation velocitiesin sodium over potassium salt concentration. Accordingly, the lower saltconcentration used for determining ESS should be the same as the saltconcentration used for determining ESSK. In conclusion, OSS is basedonly on two sedimentation rates which further simplifies theexperimental procedure.

A sodium chloride solution and/or a potassium chloride solution as usedin a method disclosed herein may in some embodiments include for exampleone or more buffer compounds. Numerous buffer compounds are used in theart and may be used to carry out a method as described in this document.Examples of buffers include, but are not limited to, solutions of saltsof phosphate, carbonate, succinate, carbonate, citrate, acetate,formate, barbiturate, oxalate, lactate, phthalate, maleate, cacodylate,borate, N-(2-acetamido)-2-amino-ethanesulfonate (also called (ACES),N-(2-hydroyethyl)-piperazine-N′-2-ethanesulfonic acid (also calledHEPES), 4-(2-hydroxyethyl)-1-piperazine-propanesulfonic acid (alsocalled HEPPS), piperazine-1,4-bis(2-ethanesulfonic acid) (also calledPIPES), (2-[tris(hydroxylmethyl)-methylamino]-1-ethansulfonic acid (alsocalled TES), 2-cyclohexyl-amino-ethansulfonic acid (also called CHES)and N-(2-acetamido)-iminodiacetate (also called ADA). Any counter ionmay be used in these salts; ammonium, sodium, and potassium may serve asillustrative examples. Further examples of buffers include, but are notlimited to, triethanolamine, diethanolamine, ethylamine, triethylamine,glycine, glycylglycine, histidine, tris(hydroxymethyl)aminomethane (alsocalled TRIS), bis-(2-hydroxyethyl)-imino-tris(hy-droxymethyl)methane(also called BIS-TRIS), and N-[Tris(hydroxy-methyl)-methyl]-glycine(also called TRICINE), to name a few. In some embodiments the pH of oneor both of the two sodium chloride solutions is adjusted to a certainvalue such as a value in the range from about 6.0 to about 8.0. If thepH value is being set to a certain value, in typical embodiments bothsodium chloride solutions are set to the same value. In some embodimentsthe pH value of the sodium chloride solutions is adjusted to a valuethat at least essentially corresponds to the physiological pH value ofarterial blood, i.e. a pH value of about 7.4. The pH value is consideredas about physiological if it is within the range of 7.35 to 4.45.

In some embodiments one or both of the first and the second solutionused, e.g. one or both of two sodium chloride solutions or a sodiumchloride solution and a potassium chloride solution, further include amonosaccharide. Many monosaccharides are known in the art. In someembodiments the monosaccharide is a hexose. As illustrative examplesglucose (also called dextrose, corn sugar or grape sugar), fructose(also called fruit sugar), galactose and mannose are named here as ahexose monosaccharide. In some embodiments a combination of any two ormore hexose monosaccharides may be used in a method or use disclosedherein. In some embodiment the monosaccharide is a pentose. Examples ofa pentose monosaccharide that may be included in a method or useinclude, but are not limited to, arabinose, ribose, and xylose, as wellas combinations thereof. In some embodiments one or both of the twosodium chloride solutions further includes a disaccharide. Examples of adisaccharide include, but are not limited to, sucrose (saccharose),lactose and maltose, as well as combinations thereof. In someembodiments a so called sugar alcohol, i.e. a derivative of amonosaccharide or a disaccharide, which carries a further hydroxyl groupinstead of the formyl or keto group. Examples of a sugar alcoholinclude, but are not limited to, sorbitol, mannitol, xylitol, lactitoland maltitol.

The mono- or disaccharide or the sugar alcohol may be present in anydesired concentration as long as the respective solution has aphysiological osmolarity. The mono- or disaccharide may advantageouslybe used to adjust the osmolarity of the respective sodium chloridesolution. The mono- or disaccharide may for example be present in thesolution in a molar amount roughly in the range from about one third orabout half, including about 0.65-fold of the amount of sodium chlorideto about 5-fold or 10-fold the amount of sodium chloride, includingequal to the amount of sodium chloride. In some embodiments sodiumchloride may be present in the sodium chloride solution in a molaramount roughly in the range from about one fifth of, including equal tothe amount of the mono- or disaccharide to about three-fold or two-fold,including 1.5-fold of the amount of the mono- or disaccharide.

In typical embodiments one or both of the first and second solutions,e.g. two sodium chloride solutions or a sodium chloride solution and apotassium chloride solution further include a polymer such as apolysaccharide or a protein. In some embodiments the polymer has atleast essentially no overall net charge, that is in a sodium chloridesolution or a potassium chloride solutions used in a method disclosedherein or included in a kit disclosed herein the polymer is neutral ifthe respective sodium chloride solution is adjusted to physiological pH(supra). Of the many known polysaccharides typically a polysaccharide isselected that is soluble in water in the desired concentration. Examplesof a suitable polysaccharide include, but are not limited to, glycogen,dextran, xanthan, and pectin, as well as combinations thereof. Intypical embodiments the polymer, such as the polysaccharide or theprotein, is present in both sodium chloride solutions in the sameconcentration. An illustrative example of a suitable protein isfibrinogen.

In some embodiments the polymer is dextran with an average molecularweight of about 40,000 Dalton or more. In some embodiments 80% or moreof the dextran in the respective sodium chloride solution(s) has anaverage molecular weight of about 40,000 Dalton or more. As anillustrative example, 90% or more of the dextran in the respectivesodium chloride solution(s) may have an average molecular weight ofabout 40,000 Dalton or more. The dextran included in a sodium chloridesolution used in a method or included in a kit described in thisdocument may have any desired molecular weight distribution and be fromany desired source. In some embodiments the dextran has a molecularweight distribution that spans over 10,000 Dalton or more, includingover 15,000 Dalton or more. In some embodiments the dextran has amolecular weight distribution in a range of about 5,000 Dalton or less,including about 2,500 Dalton or less. In some embodiments the dextranhas a molecular weight distribution in a range of about 1,000 Dalton orless. In some embodiments the dextran has a molecular weightdistribution in a range of about 2,000 Dalton or less. In someembodiments the dextran has a molecular weight distribution in a rangeof about 1,000 Dalton or less. In some embodiments at least essentiallyall of the dextran molecules in a sodium chloride solution present havea molecular weight of at least about 40,000 Dalton. In some embodimentsat least essentially all of the dextran molecules in a sodium chloridesolution present have a molecular weight of at least about 100,000Dalton.

In some embodiments the polymer is dextran with an average molecularweight of about 70,000 Dalton or more. In some embodiments 70% or moreof the dextran in the respective sodium chloride solution(s) has anaverage molecular weight of about 70,000 Dalton or more. As anillustrative example, 80% or more, including about 90% or more, of thedextran in the respective sodium chloride solution(s) may have anaverage molecular weight of about 70,000 Dalton or more. In someembodiments at least essentially all of the dextran molecules in asodium chloride solution present in an embodiment disclosed herein havea molecular weight of at least 70,000 Dalton. In one embodiment a sodiumchloride solution used/present has a molecular weight of about 70,000Da. In some embodiments a sodium chloride solution used/present has anaverage molecular weight of about 200,000 Dalton to about 500,000Dalton. If dextran is used as the polymer, it is typically included in asodium chloride solution in an amount in the range from about 1% toabout 10% (w/w), including in the range from about 1.5% to about 6%(w/w). In one embodiment the amount of dextran in a respective sodiumchloride solution is selected in the range from about 2% to about 5%. Asan illustrative example, the amount of dextran in a sodium chloridesolution may be about 4% (w/w). As a further example, the amount ofdextran in a sodium chloride solution may be about 3% (w/w).

In some embodiments the polymer is present in a sodium chloride solutionin an amount that is selected in the range from about 0.01 to about 2mmol/l. The range of the polymer may in some embodiments be from about0.05 to about 1 mmol/l. As an example, the polymer may be included in asodium chloride solution in a concentration of about 0.2 mmol/l. Thepolymer may for instance be a dextran, present in a concentration ofabout 0.2 mmol/l.

The polymer is thought to assist the aggregation of erythrocytes.Without being bound by theory it is assumed that the presence of polymermolecules in the sodium chloride solution has an inducing effect onerythrocyte aggregation once the erythrocytes have come in closeproximity to one another. It has previously been suggested that amechanism of depletion contributes to this assisting effect onaggregation albeit a bridging model has also been discussed.

In a method described herein erythrocytes from the subject are beingsuspended in a first and in a second solution as defined above.Generally about the same amount of erythrocytes per volume is beingsuspended in both solutions. Any desired container may be used forsuspending the erythrocytes. If the erythrocytes have been obtained bycentrifugation, the pellet that includes, essentially consists of, orconsists of the erythrocytes may be resuspended in the respective sodiumchloride or potassium chloride solution. Generally the suspendederythrocytes are placed in a container that allows distinguishing aclear supernatant from a suspension of erythrocytes. In this containerthe erythrocytes are being allowed to settle for a limited period oftime (see below). The container generally has a circumferential wall,which may be a lateral wall, with a wall portion that allows light toenter the container, i.e. a light incident wall portion, and a lightemerging wall portion, which is a wall portion that allows a view intothe container to an extent that a clear supernatant and a cellsuspension can be distinguished. At least one of these wall portions mayfor instance be a straight wall. In some embodiments these two wallportions are of the same material and thus both allow distinguishing asupernatant from a cell suspension. Examples of suitable material forthe light incident wall portion and the light emerging wall portioninclude, but are not limited to, glass, quartz and plastic material.Suitable plastic materials for the light incident wall portion and thelight emerging wall portion include, but are not limited to,polymethylmeacrylates (e.g. polymethyl-methacrylate (PMMA) or carbazolebased methacrylates and dimethacrylates), polystyrene, polycarbonate,and polycyclic olefins. A further illustrative example of a materialthat is additionally suitable for a wall portion that allows light topass only to a certain extent is fluoro-ethylen-propylen (FEP). In someembodiments the light incident wall portion and the light emerging wallportion are transparent or at least essentially transparent in the rangeof visible light. In one embodiment the light incident wall portion andthe light emerging wall portion are at least essentially transparent.The transmission properties of a respective wall portion may alsogradually or step-wise change from transparent to opaque, for examplefrom one end of a respective wall portion to another end.

In some embodiments a suspension of erythrocytes is prepared in the samecontainer in which the erythrocytes are afterwards being allowed tosettle. In some embodiments a suspension of erythrocytes is prepared ina container that differs from the container in which the erythrocytesare being allowed to settle subsequently. The suspension of erythrocytesis in such embodiments transferred to the container in which theerythrocytes are being allowed to settle. In some embodiments thecontainer into which erythrocytes are being transferred is a tube, whichmay have a straight circumferential wall. A respective tube may forinstance be a capillary, which may have a sealable end. Such a capillarymay have an open end and a sealed end.

Further, the erythrocytes in the suspension in the sodium chloridesolution(s) and/or potassium chloride solution(s) are being allowed tosettle for a limited period of time, i.e. they are allowed to settle fora period of time in which a suspension of erythrocytes and a supernatantform. Put differently, the period of time selected for allowing theerythrocytes to settle is short enough to avoid the formation of apellet of cells at the bottom of the respective container. The period oftime for allowing the erythrocytes to settle, which may also beaddressed as allowing the erythrocytes to sediment or allowing theerythrocytes to associate with each other, may in some embodiments beselected in a range from about 20 minutes to about 240 minutes. Theerythrocytes may for example be allowed to settle for a period of timefrom about 30 minutes to about 200 minutes. The period of time is insome embodiments about 45 minutes. In some embodiments the erythrocytesare allowed to settle for about 80 minutes. As an illustrative example,the erythrocytes may have been suspended in two saline solutions ofdifferent sodium chloride concentration, which encompass dextran andwhich have an approximately physiological osmolarity, i.e. about 260mOsm/L. The suspensions may then be left unagitated for about 80minutes, thereby allowing the erythrocytes to settle. In someembodiments the erythrocytes may be allowed to settle for about 135 orfor about 150 minutes.

As noted above, while the erythrocytes are allowed to settle, theerythrocytes aggregate, i.e. the density of erythrocytes in the solutionincreases. As a result a supernatant forms—that is, a layer of thesodium chloride or potassium chloride solution above a suspension oferythrocytes which is a layer free of erythrocytes. While theerythrocytes continue to aggregate, the size of the supernatantincreases. Since the rate of aggregation differs between the suspensionwith the high sodium chloride concentration and the suspension with thelow sodium chloride concentration, a difference in the height of thesupernatants forming as a top layer on the respective two solutions canbe observed. This difference is allowed to increase at least until aclear and significant difference can be detected. Often a duration ofabout 20 minutes is sufficient to clearly distinguish the height of thesupernatants between the two suspensions. In some embodiments apredetermined time interval is observed for allowing the two suspensionof erythrocytes to settle. In some embodiments the two suspension oferythrocytes are allowed to remain unagitated for a period from about 25minutes to about two hours, such as from about 35 minutes to about 100minutes.

In some embodiments a method disclosed herein includes carrying out acontrol measurement. The control measurement is in some embodiments ameasurement that may be carried out using erythrocytes from the samesubject that were collected at a different point in time, for exampleone or more months or one or more years before the current measurementis being carried out. In some embodiments the control measurement iscarried out with erythrocytes from another subject. Such other subjectmay be a subject with a blood pressure of known salt sensitivity and/ora patient of a known risk level of occurrence of hypertension. Saltsensitivity and/or risk level of occurrence of hypertension may forinstance be average for a respective age group. The difference in heightof supernatant between the two suspensions with erythrocytes from thesubject may be compared to the difference in height of supernatantobserved in the control measurement. In some embodiments the differencein height of supernatant between the two suspensions is converted into aratio. Such a ratio may for instance be the height of supernatantbetween the first and the second solution, or put differently, thechange in height of erythrocyte suspension (below the supernatant) ofone of the first and the second solution over the change in height oferythrocyte suspension of the other solution. In some embodiments aratio is determined of the height of supernatant of the second solutionover the height of supernatant of the first solution.

In some embodiments the difference in height of the supernatants iscompared to a predetermined reference value. In some embodiments thereference value is based on the difference between height ofsupernatants of erythrocytes of a control sample. A control sample mayfor example be a blood sample or a blood cell sample of a subject knownnot to be at elevated risk of suffering from hypertension or fromaspects of a hypertension induced disease. In some embodiments areference value is based on a control or reference value obtainedconcomitantly with the value of the sample from the subject. In someembodiments a respective control or reference value is determined at adifferent point in time, for example at a point in time earlier than themeasurement of the sample from the subject is carried out. It isunderstood that the terms ‘control’ and ‘reference’ may in someembodiments be a range of values.

In some embodiments the determined difference in height of supernatantsis regarded as increased relative to the difference in height of areference value where the obtained value is about 1.2 times or morehigher, including about 1.5 times, about two fold, about 2.5-fold, aboutthree fold, about 3.5 fold, about 4-fold, about 5-fold or more higherthan the expression level determined in a control sample. In someembodiments the determined difference in height is regarded as higherthan a control where the obtained value is increased by an amount thatis about 0.8-fold or more of the difference in height detected in acontrol sample. The determined difference in height may for example beregarded as different from the difference in height of a control if avalue is about 30%, such as about 40% or about 50% higher or more thanthe difference in height determined in a control sample. In someembodiments a difference in height is regarded as different if theobtained value is about 60%, including about 80% higher than thedifference in height in a control sample. A difference in height ofsupernatants is in some embodiments regarded as different if theobtained value is higher by about 20%, such as about 25% or more whencompared to the difference in height between supernatants of a controlsample.

A reference value may serve as a basis for a threshold value. If thedifference in height of the supernatants between a first and a secondsolution exceeds the threshold value, the subject may be diagnosed asbeing at an increased risk of developing hypertension and/or ofdeveloping a hypertension induced condition.

Population studies may also be used to select a threshold value.Receiver Operating Characteristic (“ROC”) arose from the field of signaldetection theory developed during World War II for the analysis of radarimages, and ROC analysis is often used to select a threshold able tobest distinguish a diseased subpopulation from a nondiseasedsubpopulation. A false positive in this case occurs when a person testspositive, but actually does not have the disease. A false negative, onthe other hand, occurs when the person tests negative, suggesting theperson is healthy, when it actually does have the disease. To draw a ROCcurve, the true positive rate (TPR) and false positive rate (FPR) aredetermined as the decision threshold is varied continuously. Since TPRis equivalent with sensitivity and FPR is equal to 1—specificity, theROC graph is sometimes called the sensitivity vs (1—specificity) plot. Aperfect test will have an area under the ROC curve of 1.0; a random testwill have an area of 0.5. A threshold is selected to provide anacceptable level of specificity and sensitivity.

In addition to threshold comparisons, other methods for correlatingassay results to a patient classification (occurrence or nonoccurrenceof disease, likelihood of an outcome, etc.) include decision trees, rulesets, Bayesian methods, and neural network methods. These methods canproduce probability values representing the degree to which a subjectbelongs to one classification out of a plurality of classifications.

The comparison to a threshold value, which may be a predeterminedthreshold value, can be carried out manually, semi-automatically or in afully automated manner. In some embodiments the comparison may becomputer assisted. A computer assisted comparison may employ valuesstored in a database as a reference for comparing an obtained value or adetermined amount, for example via a computer implemented algorithm.Likewise, the comparison to a reference measurement may be carried outmanually, semi-automatically or in a fully automated manner, includingin a computer assisted manner. A computer assisted comparison may relyon the storage of data, for instance in connection with determining athreshold value, on the use of computer readable media. Suitablecomputer readable media may include volatile, e.g. RAM, and/ornon-volatile, e.g. ROM and/or disk, memory, carrier waves andtransmission media such as copper wire, coaxial cable, fibre opticmedia. Exemplary carrier waves may take the form of electrical,electromagnetic or optical signals conveying digital data streams alonga local network or a publically accessible network such as the Internet.

In some embodiments a method as described above is repeated after aperiod of one or more days, such as after one or more weeks, includingone or more months, such as four, six, eight months or more. Therepeated performances of the method may be carried out independent fromone another. Hence, time intervals or combinations used may beindependently selected. In some embodiments the repeated performances ofthe method may be carried out with exactly identical settings. Forindividual instances of measurements of repeated performances of amethod described herein the composition of the first and the secondsolution may be independently selected. In some embodiments themeasurements of formation of a supernatant of an erythrocyte suspensionare carried out using the same composition of two sodium chloridesolutions or of a sodium chloride solution and a potassium chloride inthe repeated instances of carrying out the method or use.

In some embodiments where a plurality of sessions of a method disclosedherein are being carried out, in each session a plurality ofmeasurements of supernatant height differences are been carried out. Insome of these embodiments with repeated supernatant height detectionthese repeated measurements are taken within a time interval of lessthan a day such as less than three, or less than one hour.

In some embodiments a plurality of sessions of a method described hereinis carried out in order to monitor a subject's risk of developinghypertension. The measurement of supernatant height differences may insuch embodiments be repeated after a period of several months such asabout 6 or about 9 months or after a period of several years such asabout 2 years, about 5 years or about 10 years. One session may in sucha monitoring serve as a reference session. This session may be carriedout in the same way, i.e. using a pair of two sodium chloride solutionsor of a sodium chloride solution and a potassium chloride with the samecomposition as in another session. A respective reference session may bea session where a method described herein is being carried out for thefirst time.

In some embodiments a method or use disclosed herein is a method or ause for diagnosis or for stratifying the risk of disease. The term “riskstratification” as used herein generally includes identifying subjectshaving a prognosis of developing hypertension, subjects having aprognosis of exacerbation in hypertension, as well as identifyingsubjects having a low, a medium and a high risk of developinghypertension. The term includes finding subjects having a blood pressurethat is sensitive to sodium intake. The term includes finding healthysubjects whose blood pressure may rise to hypertension levels in thefuture. The term also includes finding subjects suffering fromhypertension and whose blood pressure is sensitive to sodium intake. Inthis regard a method disclosed herein may be useful in both diagnosisand treatment. Measures may for example be taken to reduce the subject'ssodium amount in the body, for example to increase sodium clearanceand/or to reduce sodium intake. Stratification may be based on theprobability (or risk) of developing or of exacerbation of hypertension.A method or use disclosed herein may also serve in stratifying theprobability of the risk of any given cardiovascular disease or the riskof any given cardiac or cardiovascular event for a subject.

A large variety of hypertension related disorders are known in the art.Hypertension is known to result in damage and/or disorders of variousorgans, in particular of the heart, the brain, the kidney and the retinaof the eye. In some embodiments a hypertension related condition is acardiovascular disease. A risk of a condition to which a method or useaccording to the invention relates (supra) includes, but is not limitedto, a risk of atherosclerosis, of coronary atherosclerosis, of vascularstenosis, of thrombosis, of peripheral vascular disease, includingperipheral artery disease, of stroke, of haemorrhagic stroke, ofischemic stroke, of ischemic heart disease, of congestive heart failure,of Angina pectoris, myocardial infarction of ventricular arrhythmias, ofmyocardial ischemia, of coronary heart diseases, of acute coronarysyndrome, of (acute) myocardial infarct, of cardiac insufficiency, ofangina pectoris, of renal artery stenosis or of renal nephrosclerosis.“Ischemic heart disease” or myocardial ischemia as used herein isunderstood as referring to a disease characterized by reduced bloodsupply to the heart muscle, usually due to coronary artery disease(atherosclerosis of the coronary arteries). A “stenosis” as used herein,is defined as an abnormal narrowing in a blood vessel or other tubularorgan or structure. “Cardiac insufficiency” as used herein refers to asan acute or chronic inability of the heart to supply sufficient blood tothe tissue, and as a result, oxygen, to guarantee tissue metabolism atrest and under stress. Clinically, cardiac insufficiency is present,when typical symptoms (dyspnea, fatigue, liquidity retention) exist, thecause of which is based on a cardiac dysfunction within the meaning of asystolic or diastolic dysfunction.

In some embodiments where an increased difference in height between thesupernatants is detected, the subject's blood pressure is determined. Itmay be decided to determine the subject's blood pressure at certain timeintervals if the difference in height between the supernatants is foundto be elevated. The subject's blood pressure may be monitored, forinstance on an annual, on a monthly or on a weekly basis. Blood pressuremay be determined according to any desired method at any one or morearteries of a subject. In typical embodiments peripheral blood pressureis determined, for example at one or more limbs of the subject. In someembodiments arterial blood force, i.e. the pressure applied by blood tothe arterial wall, is determined. In some embodiments a blood pressuremeasurement of a human may for instance be carried out in the form ofthe indirect method first described by von Riva Rocci Recklinghaus. Inthis method an inflatable cuff is placed on the middle third of theupper arm and the pressure within the cuff is quickly raised up tocomplete cessation of circulation below the cuff, typically by inflatingthe cuff to a predetermined level. An electronic blood pressuremeasuring device or a reservoir of mercury at the end of a verticalglass column, in combination with listening to the artery just below thecuff with a stethoscope, may be used to determine the blood pressurevalue. In some embodiments an automatic, typically non-invasive, bloodpressure gauge based on e.g. a pressure sensor, for example incombination with a press mechanism and/or a flow sensor and asphygmomanometer, may be used.

A respective blood pressure measurement may be repeated. Such ameasurement may be a measurement of the systolic blood pressure, ameasurement of the diastolic blood pressure or both. Where a pluralityof blood pressure measurements is carried out, the blood pressuremeasurements may be taken, at least substantially, after unitary timeintervals or after varying time intervals. In some embodiments repeateddetermination of the subject's blood pressure is carried out atintervals of a certain, e.g. predetermined, length. As an illustrativeexample, the time interval between repeated measurements of the bloodpressure may in some embodiments be selected in the range from about 6hours to about 12 months, such as from about a day to about 6 months orfrom about a week to about 2 months.

A kit as described herein may include, including consist of, a pair oftubes and a container that includes a sodium chloride solution. Thesodium chloride solution may further include a mono- or disaccharidesuch as glucose as well as a polysaccharide. In some embodiments the kitincludes two containers that include a sodium chloride solution.Typically both containers with sodium chloride solution include at leastessentially the same components with the only difference being theconcentration of sodium chloride. In some embodiments the kit includestwo containers, one container including a sodium chloride solution, andone container including a potassium chloride solution. Typically bothcontainers with sodium chloride solution or with a sodium chloridesolution and a potassium chloride solution include at least essentiallythe same components with the only difference being the concentration ofsodium chloride or the presence of potassium chloride instead of sodiumchloride. The kit may be used for carrying out a method as describedabove. The kit may be for prognosis, diagnosis and/or riskstratification of disease. The first container may be used for a sessionof the method described herein that serves as a control measurement.

Additional objects, advantages, and features of this disclosure willbecome apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting. Thus,it should be understood that although the present disclosure isspecifically disclosed by exemplary embodiments and optional features,modification and variation of the disclosures embodied therein hereindisclosed may be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis disclosure.

EXAMPLES

The following example serves in illustrating an embodiment of carryingout a method as disclosed herein. Furthermore it is illustrated how theinteraction of erythrocytes (red blood cells; RBC) and vascularendothelium can be assessed using atomic force microscopy (AFM).

I: Salt-Blood-Test (SBT)

A. Protocol of carrying out the method based on venous puncture

-   -   1. 1 ml of blood (for double-measurement) is drawn by venous        puncture using EDTA-K or heparinized monovettes (Sarstedt        Company, Sarstedt, Germany).    -   2. Blood is transferred into 2 ml plastic vials (Eppendorf AG,        Hamburg, Germany) and centrifuged for 5 minutes at 1800×g.    -   3. Plasma and buffy coat are removed and erythrocytes washed 2        times in 30 ml of HEPES-buffered (10 mM        HEPES=2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethane sulfonic acid)        sodium chloride (150 mM NaCl) including 1% bovine serum albumin        (PAA Clone, Coelbe, Germany).    -   4. 80 μl of washed erythrocytes (ERY) are suspended in 120 μl        NaCl solution (150 mM NaCl, Roth Company, Karlsruhe) containing        3% dextrane (Sigma 44886, MW: 70,000 Dalton).    -   5. 50 mM sucrose (Sigma 0389) is added to low NaCl solutions        (125 mM) for maintaining constant osmolality (in relation to the        150 mM NaCl).    -   6. Hematocrit capillary tubes (Safecap P75-2000; length: 75 mm;        Scholz Company, Neubiberg, Germany) are filled by capillary        forces with the respective erythrocyte solutions (150 mM NaCl        and 125 mM NaCl, as selected).    -   7. The hematocrit capillary tubes, closed on the lower end, are        put on stands in an upright position.    -   8. Sedimentation rates of red blood cells are measured between        60 and 120 minutes and ESS (Erythrocyte Salt Sensitivity) is        calculated (from the 75 minutes) values as follows:

ESS=L ₁₅₀ /L ₁₂₅ (as used in FIGS. 19, 20, 22)

-   -    or expressed as a percentage:

ESS (%)=[1−(L ₄₂₅ /L ₁₅₀)]×100 (as used in FIGS. 5,6)

B: Protocol of carrying out the method based on capillary blood fromfinger tip

-   -   1. 200 μl of capillary blood is taken by puncture of the        fingertip using hematocrit capillaries (3.75 IU        Na-heparin/capillary; Hirschmann Laborgeräte, Eberstadt,        Germany).    -   2. Blood is transferred into 2 ml plastic vials (Eppendorf AG,        Hamburg, Germany) and centrifuged for 5 minutes at 1800×g.    -   3. Plasma and buffy coat are removed and erythrocytes washed        twice in 1400 μl of HEPES-buffered (10 mM        HEPES=2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethane sulfonic acid)        sodium chloride (150 mM NaCl) including 1% bovine serum albumin        (PAA Clone, Coelbe, Germany).    -   4. In order to obtain an in vitro hematocrit of 40%, 35 μl of        washed erythrocytes are suspended in 52.5 μl NaCl solutions (125        and 150 mM NaCl, respectively; Carl Roth Company, Karlsruhe,        Germany; each solution contains 3% dextrane; Sigma 4486, MW:        70,000) and mixed for 30 sec on a shaker (750 rounds per        minute).    -   5. 50 mM sucrose (Sigma 0389) is added to low NaCl solutions        (125 mM) for maintaining constant osmolality (in relation to the        150 mM NaCl).    -   6. Hematocrit capillary tubes (Safecap P75-2000; length: 75 mm;        Scholz Company, Neubiberg, Germany) are filled by capillary        forces with the two respective erythrocyte solutions (as        described above).    -   7. The hematocrit capillary tubes, closed on the lower end, are        put on stands in an upright position.    -   8. Sedimentation rates are measured between 60 and 120 minutes        and ESS (Erythrocyte Salt Sensitivity) is calculated (from the        75 minutes) values as follows:

ESS=L ₁₅₀ /L ₁₂₅ (as used in FIGS. 19, 20, and 22)

ESS can also be expressed as a percentage, calculated (from the 75minutes) as:

ESS (%)=[1−(L ₁₂₅ /L ₁₅₀)]×100 (as used in FIG. 5 and FIG. 6)

The test was carried out on blood samples from 12 test persons (cf. FIG.6).

In contrast to the standard erythrocyte sedimentation rate (ESR), whichis performed in whole blood and strongly dependent on anyinflammation-relevant proteins in the plasma phase, the sedimentationrate used for ESS measurements is independent of any proteins, butstrongly dependents on the NaCl concentration of the electrolytesolution in which the erythrocytes have been suspended. In particular,sedimentation rate depends on the RBC zeta potential and, thus, on thered blood cell glycocalyx. Without being bound by theory, since Na⁺neutralizes the negative charges of the red blood cell surface, it canbe assumed that sedimentation rate reflects the Na⁺ buffer capacity ofthe red blood cell glycocalyx. Dextran is typically to be added to allsolutions for aggregation to occur. In pilot studies, the inventorsfound that a particular suitable molecular mass of the dextran is 70,000D, and an advantageous concentration is 3%. FIG. 3 shows the timedependence of the red blood cell sedimentation rate. Initialsedimentation rates are quite linear, but they saturate with progressionof time. This is due to the fact that a a hematocrit of 0.40 was used sothat after extended time periods (hours), virtually all red blood cells,not only the large red blood cell aggregates, concentrate in the lowerpart of the tube. Therefore, the 60-min values were chosen for analysis.FIG. 4 shows the dependence of the erythrocyte sedimentation on NaClconcentration. Ion strength, and in particular the cation Na⁺,determines sedimentation velocity.

FIG. 5 displays two representative measurements. From the respectivesupernatants, ESS could be calculated. The ESS of FIG. 5A was 2.2,indicating that RBC sedimentation rate was 2.2 times larger in 150 mMNa⁺ than the sedimentation rate in 125 mM Na⁺. The ESS of FIG. 5B was5.8, indicating that RBC sedimentation rate was 5.8 times larger in 150mM Na⁺ than that in 125 mM Na⁺. Thus, ESS of blood used in FIG. 5B wasmore than twice as high as the ESS of blood used in FIG. 5A. In a seriesof control experiments, the heparan sulfate residues of the RBCglycocalyx were enzymatically removed by heparinase I incubation(Oberleithner, H., Pflügers Arch. (2013) doi:10.1007/s00424-013-1288-y),and then, ESS was measured.

In six experiments, ESS increased only by about 13% (from 2.6±0.12 to3.0±0.14), indicating that not only heparin sulfate residues but alsoother negatively charged (and heparinase-insensitive) components of theglycocalyx contribute to the sodium-dependent sedimentation rate. FIG.22 depicts ESS measured in 61 healthy volunteers of similar age. Thefrequency distribution indicates two ESS peaks, one at about 3 andanother one at about 5. FIG. 19 shows these data in more detail.Forty-six percent (28 out of 61) of the study participants exhibited anESS of at least 20% below average. Twenty-eight percent (17 out of 61)of the study participants exhibited an ESS of at least 20% aboveaverage. No significant gender difference of the respective ESS wasobserved. It is worth mentioning that the salt blood test has beenperformed in all experiments at a fixed hematocrit (0.40). Takentogether, the data indicate that there is a wide range of ESS (from 2 to8) within the normal population. It should be mentioned that at leastfive individuals (white bars in FIG. 19) were on low-salt diets (for noobvious reason, they indicated this explicitly in a questionnaire).Remarkably, the ESS of all five of them was found in the “weaklysalt-sensitive” group.

II: Salt-Blood-Test by Measuring the Na+ Over K+ Ratio (ESSK)

Protocol based on capillary blood from the fingertip (excluding acentrifugation step)

-   -   1. 200 μl of capillary blood is taken by puncture of the        fingertip using hematocrit capillaries (3.75 IU        Na-heparin/capillary; Hirschmann Laborgeräte, Eberstadt,        Germany).    -   2. Blood is transferred into a 500 μl plastic vial (Eppendorf        AG, Hamburg, Germany) and Erythrocytes allowed to sediment for        90 minutes by gravidy at room temperature.    -   3. Plasma and buffy coat are removed and the erythrocyte        concentrate (about 100 μl) at the bottom of the plastic vial        taken up by a pipette and then 40 μl of erythrocyte concentrate        pipetted into two (empty) 500 μl vials (40 μl each).    -   4. In order to obtain an in vitro hematocrit of 40%, 60 μl of a        NaCl or a KCl solution (125 mM; each solution contains 3%        dextrane; Sigma 4486, MW: 70,000) was added and mixed.    -   5. 50 mM sucrose (Sigma 0389) has been added before for        maintaining normal osmolality (about 300 mosmol/l).    -   6. Hematocrit capillary tubes (Safecap P75-2000; length: 75 mm;        Scholz Company, Neubiberg, Germany) are filled by capillary        forces with the two respective erythrocyte solutions (as        described above).    -   7. The hematocrit capillary tubes, closed on the lower end, are        put on stands in an upright position.    -   8. Sedimentation rates are measured between 60 and 120 minutes        and ESS (Erythrocyte Salt Sensitivity) is calculated (from the        60 minutes) values as follows:

ESSK=L _(125 Na) ⁺ /L _(125 K) ⁺

III: Analysis of the Interaction of Erythrocytes and VascularEndothelium Using Atomic Force Microscopy

Protocol of Carrying Out the Method:

-   -   1. Endothelial monolayers (Eahy629 cell line [Oberleithner, H.,        et al., Proc Natl Acad Sci USA (2007) 104, 16281-16286;        Oberleithner, H., et al., Proc Natl Acad Sci U S A (2009) 106,        2829-2834] cultured on the bottom of 75 cm² culture flasks) are        incubated at 37° C. with whole blood (5 ml) taken from        volunteers over a time period of 18 to 22 hours. The flasks are        continuously agitated (1 to 5 RPM, 7 degree angle) so that the        RBC can physically interact with the surface of the endothelial        cell layer.    -   2. The experiment is performed on intact endothelium and on        endothelium that has been pretreated with heparinase (heparinase        I, Sigma Aldrich H 2519, Taufkirchen, Germany) in order to        remove the negatively charged heparan sulphate residues from the        endothelial glycocalyx.    -   3. After this procedure the RBC are harvested, seeded on        poly-l-lysine coated glass cover slips, fixed with 0.1% fixative        (glutaraldehyde in HEPES buffer) and imaged by using an atomic        force microscope.

AFM Imaging:

Imaging of the RBC surface was performed with methods basicallydescribed previously [Oberleithner, H., et al.: Nanoarchitecture ofplasma membrane visualized with atomic force microscopy. Methods inPharmacology: Ion channel localization methods and protocols. Edited by:A. Lopatin and C. G. Nichols. 2001. Humana Press Inc.; Oberleithner, H.,et al., J Membr Biol (2003) 196, 163-172; Oberleithner, H., et al.,Hypertension (2004) 43, 952-956; Schneider, S. W., et al., Methods MolBiol (2004) 242, 255-279]. In order to image the glycocalyx of RBC thefollowing protocol was used: A glass coverslip (diameter 15 mm), coveredwith RBC was mounted on the stage of the AFM and individual RBC wereimaged in buffered electrolyte solution. Then, 2 U/ml of heparinase wereadded to the RBC. The RBC surface was continuously imaged over 30minutes while the enzyme was active. Images were stored on the computer.After the experiment the respective images before and after applicationof heparinase were electronically substracted. This results in aso-called net-image that shows the glycocalyx (i.e. the enzyme-sensitivepart of the RBC surface). Since the AFM generates 3-D images, height andvolume of the images can be quantitatively evaluated. This complex andhighly sophisticated method that needs in-depth AFM expertise and thatis based on expensive AFM technology cannot be used for any clinicalstudy.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present specification, including definitions, willcontrol. Applicants reserve the right to physically incorporate intothis application any and all materials and information from any sucharticles, patents, patent applications, or other physical and electronicdocuments.

The listing or discussion of a previously published document in thisspecification should not necessarily be taken as an acknowledgement thatthe document is part of the state of the art or is common generalknowledge.

One skilled in the art would readily appreciate that the presentmethods, kits and uses are well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those inherenttherein. Further, it will be readily apparent to one skilled in the artthat varying substitutions and modifications may be made to theinvention disclosed herein without departing from the scope and spiritof the invention. The compositions, methods, procedures, treatments,molecules and specific compounds described herein are presentlyrepresentative of preferred embodiments are exemplary and are notintended as limitations on the scope of the invention. Changes thereinand other uses will occur to those skilled in the art which areencompassed within the spirit of the invention are defined by the scopeof the claims. The listing or discussion of a previously publisheddocument in this specification should not necessarily be taken as anacknowledgement that the document is part of the state of the art or iscommon general knowledge.

The methods, uses and kits illustratively described herein may suitablybe practiced and applied in the absence of any element or elements,limitation or limitations, not specifically disclosed herein. Thus, forexample, the terms “comprising”, “including,” containing”, etc. shall beread expansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereon It is recognized that variousmodifications are possible. Thus, it should be understood that althoughthe present disclosure has been specifically disclosed by exemplaryembodiments and optional features, modification and variation of thedisclosures embodied therein herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this disclosure.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods, uses and kits. This includesthe generic description of the methods, uses and kits with a proviso ornegative limitation removing any subject matter from the genus,regardless of whether or not the excised material is specificallyrecited herein.

Other embodiments are set forth within the following claims. Inaddition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

1. An in vitro method of analyzing whether the blood pressure of asubject is sensitive to sodium intake, the method comprising: (a)suspending erythrocytes of the subject in one or more pairs of a firstsolution of about physiological osmolality and a second solution ofabout physiological osmolality, each first solution and each secondsolution comprising at least one of sodium chloride and potassiumchloride,  wherein for each pair of first and second solution the firstsolution comprises sodium chloride, and the second solution comprisessodium chloride or potassium chloride,  wherein a second solutioncomprising sodium chloride has a sodium chloride concentration that isat least about 25 mM higher than the sodium chloride concentration ofthe first solution, and  wherein a second solution comprising potassiumchloride has a concentration of potassium chloride that is of at leastessentially about the same value as the concentration of sodium chloridein the first solution; (b) allowing the suspended erythrocytes in thefirst and in the second solution of at least one pair of first andsecond solution to settle for a period of time sufficient to allow theformation of a supernatant; and (c) detecting the difference in heightof the supernatant between the first and the second solution of the atleast one pair of first and second solution,  wherein an increaseddifference in height of the supernatants of the at least one pair offirst and second solution, relative to a threshold value, indicates thatthe blood pressure of the subject is sensitive to sodium intake.
 2. Themethod of claim 1, wherein the method is a method of assessing thesusceptibility of a subject to develop hypertension, and wherein anincreased difference in height of the supernatants relative to athreshold value, indicates an increased susceptibility of the subject todevelop hypertension.
 3. The method of claim 1, wherein each of thefirst and the second solution of a pair of a first solution and a secondsolution has a concentration of sodium chloride or potassium chloridethat is about 100 mM or more.
 4. The method of claim 1, wherein for oneor more pairs of a first and a second solution of about physiologicalosmolality, a second solution comprising potassium chloride has aconcentration of potassium chloride that is about 25 mM or more higherthan the sodium chloride concentration of the first solution.
 5. Themethod of claim 1, wherein the first and the second solution of a pairof a first solution and a second solution further comprise apolysaccharide.
 6. The method of claim 5, wherein the polysaccharide isdextran.
 7. The method of claim 5, wherein the polysaccharide has anaverage molecular weight of about 70,000 Da.
 8. The method of claim 5,wherein the polysaccharide is comprised in the first and the secondsolution in an amount of about 3%.
 9. The method of claim 1, wherein atleast one of the first and the second solution of a pair of a firstsolution and a second solution comprises a monosaccharide or adisaccharide.
 10. The method of claim 9, wherein the disaccharide issucrose.
 11. The method of claim 1, wherein the threshold value is basedon the difference in height of the supernatant between a correspondingfirst and a corresponding second solution of erythrocytes of a controlsample.
 12. The method of claim 1, wherein the period of time forallowing the suspended erythrocytes to settle is selected in the rangefrom about 45 to about 120 minutes.
 13. The method of claim 1,comprising suspending erythrocytes of the subject in a first and asecond pair of solutions, each pair of solutions comprising a firstsolution and a second solution, wherein for the first pair of solutions,both the first solution and the second solution comprise sodiumchloride, and for the second pair of solutions the first solutioncomprises sodium chloride, and the second solution comprises potassiumchloride, and wherein the concentration of sodium chloride in the firstsolution of the first pair of solutions is at least about the same asthe concentration of potassium chloride in the second solution of thesecond pair of solutions.
 14. A kit of parts comprising a pair of tubesand one or more pairs of a first and a second container, a firstcontainer comprising a first solution and a second container comprisinga second solution, each first solution and each second solutioncomprising at least one of sodium chloride and potassium chloride,wherein for each pair of a first and a second container the firstsolution comprises sodium chloride, and the second solution comprisessodium chloride or potassium chloride, wherein a second solutioncomprising sodium chloride has a sodium chloride concentration that ishigher than the sodium chloride concentration of the first solution, andwherein a second solution comprising potassium chloride has aconcentration of potassium chloride that is of at least about the samevalue as the concentration of sodium chloride in the first solution. 15.The kit of claim 14, wherein for a pair of a first and a secondcontainer, a second solution comprising sodium chloride has a sodiumchloride concentration that is at least about 25 mM higher than thesodium chloride concentration of the first solution.
 16. The kit ofclaim 14, wherein each of the first and the second solution is of aboutphysiological osmolality.
 17. The kit of claim 14, wherein each of thefirst and the second solution has a concentration of sodium chloride orpotassium chloride of about 100 mM or more.
 18. The kit of claim 14,wherein each of the first and the second solution further comprises apolysaccharide.
 19. The kit of claim 14, wherein for a pair of a firstand a second container at least one of the first and the second solutioncomprises a monosaccharide or a disaccharide.
 20. The kit of claim 14,wherein each of the tubes of the pair of tubes is a capillary, thecapillary having an open end and a sealable end.